Novel methods and compositions to evaluate and determine inactivation of hazardous biological material

ABSTRACT

Novel time and temperature integrator (TTI) assays, kits containing the components of the assays, and the novel components for those assays are provided herein. These novel TTI assays evaluate and/or determine the inactivation of biological material in/on a sample by quantifying the degradation of DNA using qPCR. The sample can be a food product (e.g., fruits, vegetables, meat from animals, or eggs) while the item can be any object (e.g., medical equipment, especially reusable medical equipment) for which one needs to determine that the amount of inactivation of specific hazardous biological material on the object or in a sample is at or below a pre-determined amount.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. patent application Ser. No.14/917,830 filed Mar. 9, 2016, which itself is a National PhaseApplication filed under 35 U.S.C. § 371 as a national stage ofPCT/US2014/054749, filed on Sep. 9, 2014 and claims priority from bothU.S. Provisional Patent Application Ser. No. 61/876,425 filed Sep. 11,2013 and U.S. Provisional Patent Application Ser. No. 61/969,465 filedMar. 24, 2014, all of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to assays to evaluate and/or determine theinactivation of biological material in/on a sample by quantifying thelevel of integrity, or alternatively, the degradation of, DNA and thematerials necessary to perform the assays. The sample can be a foodproduct (e.g., fruits, vegetables, meat from animals, or eggs) while theitem can be any object (e.g., medical equipment, especially reusablemedical equipment) for which one needs to determine that the amount ofinactivation of specific hazardous biological material on the object orin a sample is at or below a pre-determined amount. Non-limitingexamples of hazardous biological material are toxins, viruses,parasites, fungi, bacteria, spores (bacterial, fungal or parasitical)and cancer cells. This invention further relates to the tools used inthe assays. One assay uses quantitative PCR and polynucleotide primersto assay the fragmentation of mitochondrial DNA found intrinsically inthe food matrix. Another assay examines the size and fragmentation oftotal DNA present in the food matrix using any device which measures DNAfragment size globally. A third assay involves use of an extrinsicsource of mitochondrial DNA added to the processing run in a recoverablecontainer.

SEQUENCE LISTING

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file name“SequenceListing”, created on Apr. 24, 2020, and having a size of 6kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

DESCRIPTION OF THE PRIOR ART

In order to render food products (both processed and fresh foodproducts) safe and to prevent them from spoiling under the usualconditions of storage, some form of commercial sterilization isrequired. Control of food-borne bacterial pathogens such as Salmonellaspp., Shigella spp., pathogenic Escherichia coli, Campylobacter spp.,and Yersinia spp. has traditionally been achieved using heat treatmentssuch as pasteurization or pressure sterilization. Heat treatments higherthan pasteurization are used to provide Clostridium botulinum safety inlow-acid canned foods and canned cured meats. Low-acid products have apH of approximately 4.6 and above and include most meat and marineproducts, corn, peas, lima beans, asparagus and spinach. More recently,inhibition of surviving Clostridium botulinum spores has been achievedby treating food with NaNO₂ and/or potassium sorbate. Failure toadequately inactivate Clostridium botulinum spores can result in theproduction of neurotoxins by the bacteria. Of course, failure to destroyother disease causing bacteria (e.g., Salmonella spp., Shigella spp.,pathogenic E. coli, Campylobacter spp., and Yersinia spp.) in foodproducts can result in food poisoning and severe disease after ingestingthe contaminated food products. In addition, butchered meats and raweggs can become contaminated with pathogenic bacteria during thehandling and/or processing of the meats and eggs. It is vital to reduceor eliminate the bacteria on the surface of these items so that theworkers and people that consume the food are not sickened by thebacteria.

Food spoilage and food poisoning from contamination of food by bacteriais a major problem throughout the world, including developed countriessuch as the United States, Canada, Japan, U.K., France, Germany, andRussia. In the U.S. alone, illness from food-borne bacteria costsseveral billion dollars annually in morbidity and mortality.Gram-positive and Gram-negative food-borne bacteria account for many ofthe pathogens causing food poisoning.

Traditional safety guidelines have traditionally used destruction ofbacterial surrogates as indicators of processing safety and efficacy.Culturing samples of food or items for viability of bacteria and/orspores is a current method for assaying the destruction of bacterialsurrogates. Two bacterial surrogates are Geobacillus stearothermophilusand Bacillus subtilis spores which are placed in or on samples prior toinactivation and which are cultured after treatment to determine if thebacteria and/or the bacterial spores have been inactivated. Problemswith the culture approach include tracking and recovering indicatorspores and/or bacteria, excessive time required to culture (48 hours ormore), and molecular methods unable to differentiate between live anddead surrogates. Other devices and methods have been created to assayfor live or killed bacteria and bacterial spores in food and devicesusing various culturing conditions (e.g., US Pub. 2007-0249040; U.S.Pat. Nos. 5,486,459; 5,830,683; 5,989,852; and WO 1995/008639), yetthese methods and devices lack the ability to timely and accuratelydetermine the amount of reduction of viable hazardous biologicalmaterials in or on a food or item.

To overcome the limitation of the long incubation times required for theproliferation of microbes and/or bacterial spores, time and temperatureintegrator (TTI) technologies have been developed. An example of suchtechnologies is the application of enzymes derived from microbes thatcan naturally tolerate high temperatures and need such high temperaturesto proliferate. For example, α-amylase from Bacillus licheniformis(Cordt, et al., J. Chem. Tech. and Biotech. 59:193-199 (1994)), lipase(U.S. Pat. No. 4,284,719), and β-glucosidase (Adams and Langley, FoodChemistry, 62:65-68 (1998)) have been used as TTIs. These enzymes can beproduced in high concentrations using molecular biology techniques. Thepure enzyme is then encapsulated in plastic tubing with 1 mm diameter,and the ends are sealed by melting. The encapsulated enzyme can beincorporated in a food matrix and recovered after thermal treatment(e.g., pasteurization) of the food matrix is applied. The activity ofthe post-thermal treated, heat resistant enzyme is determined andcorrelated with the effectiveness of the heating step. This rapid methodallows for evaluation of thermal treatments uniformity andeffectiveness. However, production of enzymatic TTI is complex. Also,TTI's enzymatic activity is difficult to maintain during long storageperiods and may vary within multiple production batches.

Alternative approaches to culturing bacteria and enzymatic TTI, includeassays for bacterial DNA or mRNA in samples of food using PCR andPCR-related techniques. See, e.g., U.S. Patent App. Pub. 2010-0167956 inwhich polynucleotide probes specific for E. coli are placed on a chipfor assaying for the presence of E. coli; and U.S. Patent App. Pub.2012-0288864 in which S. enterica, a food-borne pathogen, is detected byPCR using primers specific for an S. enterica gene.

The effect of high temperature on DNA degradation is well described.Above 100° C. denaturation, depurination, deamination and loss ofsecondary structure occurs (Gryson, N., Anal. Bioanal. Chem.396:2003-2022 (2010)). Although autoclaving a foodstuff at 121° C. forfifteen minutes does not destroy all DNA available for PCR (Lipp, etal., J. AOAC Int. 82(4):923-928 (1999)), recovery of reduced DNAconcentrations via quantitative PCR (qPCR) from cornmeal and watercooked for sixty minutes at 100° C. have been reported (Murray, et al.,J. of Agric. & Food Chem. 55:2231-2239 (2007)). Increased Ct (thresholdcycles) values occurred in DNA from heat-treated corn grits and cornflour when compared to untreated corn and resulted in distortions ofqPCR assays for detection of genetically modified organisms (GMO)(Moreano, et al., J. of Agric. & Food Chem. 53:9971-9979 (2005)).

However, these prior art methods for detecting bacteria via PCR areunable to determine if the bacteria or their spores are still viable ornot because PCR often is able to detect a certain fragment of DNA fromthe bacteria or spores, despite their death or inactivation. In a studyby Stam (Doctoral Thesis, NCSU Food Science; Raleigh, N.C. (2008),available at http://www.lib.ncsu.edu/resolver/1840.16/3192), Clostridiumsporogenes spores were heat-treated to 121° C. in two minutes intervalsfor eighteen minutes, and bacterial DNA degradation over time wasdetermined. It was noted that heat-treating the spores for only twominutes resulted in the absence of DNA bands using agarose gelelectrophoresis. However, the autoclaved spore DNA was still detectableby qPCR, having a reduced Ct value of 35 compared to a Ct of 12 forviable spores (Stam (2008)). Therefore, bacterial or spore DNA isdegraded but still detectable by qPCR, when using thermal processingtechniques such as heat or microwave suitable for preserving vegetablesand fruits. Obviously, the repercussions of being wrong about viabilityof biological material in or on food or other items are serious andcould result in serious morbidity and possible mortality in humans. Itcan also lead to false positives, which have a negative financial impacton the food industry. Also, excessively heating food matrices to destroybacterial or bacterial spores DNA is also problematic because thequality of the texture, flavor and appearance of the finish good isreduced.

In addition to food substances, many other items need to be renderedsterile or have a reduction in the viability of biological material onthe items prior to usage. For example, reusable medical and dentaldevices (such as, but not limited to, endoscopes, catheters, sponges,clamps, scalpels, drills, and suction tubes) need to be cleaned(biological material inactivated) after being used on one subject, priorto use on another subject. Biological material present on robust medicalequipment is often inactivated by subjecting the contaminated equipmentto high temperatures and pressure via a steam autoclave. While suchinactivation methods are very effective for more durable medicalinstruments, advanced medical instruments formed of rubber and plasticcomponents with adhesives are delicate and wholly unsuited to the hightemperatures and pressures associated with a conventional steamautoclave. Thus, some steam autoclaves have been modified to operateunder low pressure cycling programs to increase the rate of steampenetration into the medical devices or associated packages of medicaldevices undergoing cleaning. However, highly complex instruments whichare often formed and assembled with very precise dimensions, closeassembly tolerances, and sensitive optical components, such asendoscopes, may be destroyed or have their useful lives severelycurtailed by harsh inactivation methods employing high temperatures andhigh or low pressures. Endoscopes, in particular, present problems inthat such devices typically have numerous exterior crevices and interiorlumens which can harbor microbes and thus be difficult to clean usingordinary techniques. The employment of a fast-acting yet gentleinactivation method is desirable for reprocessing sensitive instruments.Other medical or dental instruments which comprise lumens are also inneed of methods of cleaning which employ an effective reprocessingsystem which will not harm sensitive components and materials. Further,the need exists for a reprocessing system having a shorter reprocessingcycle time. Regardless of how these devices are cleaned, failure toinactivate a substantial proportion of the biological material that maybe present on the item after usage could result in the dangerous illnessin the next subject on which the item is used.

The Food Safety Modernization Act (FSMA) mandates that companiesdocument risk-based preventive controls for all pre-requisite programsas part of their Food Safety program. The Food and Drug Administrationproposed guidelines under FSMA include the application of preventionstandards to sanitation and environmental controls and monitoring. Forexample, one of the modifications under consideration involvessterilization of food packaging containers prior to filling thecontainers. Glass jars used for packaging of finished acidified and acidfoods for the retail market are currently rinse with hot water, filledwith the product, and subjected to a validated pasteurization step,often considered as a critical control point to render the finished foodsafe for consumption. New guidelines would require such containers to besubjected to a sterilization treatment prior to filling. Thesterilization step for glass containers could be applied as apasteurization, ultraviolet light, high pressure, or radiationtreatment. The effectiveness of such treatments to eradicate pathogensof public health significance would have to be demonstrated after thetreatments are applied.

As such, there remains a need for one or more assays that can evaluateand/or determine the amount of inactivation of biological material on/infood and/or an object in a timely and accurate manner. There is also aneed for assays that can evaluate inactivation protocols and forevaluating deviations in processing to reduce the amount of viablebiological material in/on items. The assays described herein usequantitative PCR. PCR and real-time PCR are well-known laboratorytechniques and are accepted by AOAC International for clinical detectionassays, including assays to detect BRCA1 and BRCA2 mutations.

Mitochondrial DNA (mtDNA) is used as identifiers in many scientificdisciplines. They have been adopted for barcoding almost all groups ofhigher animals (http://www.barcoding.si.edu/). MtDNA is also used inhuman typing for forensic analysis (Hopwood, et al., Int. J. Legal Med.108(5):237-243 (1996); Andreasson, et al., Biotechniques 33(2):402-411(2002); Budowle, et al., Annu. Rev. Genomics Hum. Genet. 4:119-141(2003)) using tissues such as bones, teeth, and hair shafts for DNAextraction. MtDNA primers or probes have been developed for sourcetracking fecal contaminates in wastewater influents and effluents usingmultiplex qPCR (Caldwell, et al., Environ. Sci. & Technol., 41:3277-83(2007); Caldwell and Levine, J. Microbiol. Methods 77:17-22 (2009);Caldwell, et al., “Mitochondrial DNA as source tracking markers of fecalcontamination”, In Microbial Source Tracking: Methods, Applications, andCase Studies, eds. Harwood, Hagedorn and Blanch (Springer Science andBusiness Media, NY) 229-250 (2011)). In the food industry, PCR-basedmtDNA analyses are used in the authentication of food, and to tracecontamination of other animals in the food products (Meyer and Candrian,Lebensm.-Wiss. u.-Technol. 29:1-9 (1996); Lahiff, et al., Mol. CellProbes 15(1):27-35 (2001); Zhang, et al., Food Control 18:1149-1158(2007); Fujimura, et al., Biosci. Biotechnol. Biochem. 72:909-913(2008)). The development of those molecular tools improved themonitoring of food quality by preventing fraudulent description of foodcontent, and identifying adulterants.

SUMMARY OF THE INVENTION

It is an object of this invention to have a synthetic polynucleotidewhich has a nucleic acid sequence that is a consensus sequence to aportion of a gene that is highly conserved in plants and/or animals. Itis a further object of this invention that this synthetic polynucleotideis between approximately 80 bp and approximately 250 bp long. It is afurther object of this invention that this synthetic polynucleotide isbetween approximately 100 bp and approximately 200 bp long. It is afurther object of this invention that this synthetic polynucleotide isbetween approximately 125 bp and approximately 175 bp long. It is afurther object of this invention that the highly conserved gene be amitochondrial gene. It is a further object of this invention that themitochondrial gene be atp1. It is a further object of this inventionthat the sequence of this synthetic polynucleotide be at least 95%identical to the sequence of a portion of atp1. It is another object ofthis invention that this synthetic polynucleotide has the sequenceselected from the group consisting of SEQ ID NO: 14, 15, 16, and 17, ora sequence that is the reverse complement thereof.

It is an object of this invention to have a synthesized polynucleotidewhich a consensus sequence to a portion of a gene that is highlyconserved in plants and/or animals. It is a further object of thisinvention that the highly conserved gene be a mitochondrial gene. It isa further object of this invention that the mitochondrial gene be atp1.It is a further object of this invention that the sequence of thissynthetic polynucleotide be at least 95% identical to the sequence of aportion of atp1. It is another object of this invention that thepolynucleotide has a sequence selected from the group consisting of SEQID NO: 1, 2, 3, 4, 5, 6, 7, and 8.

It is an object of this invention to have a composition useful fordetermining the integrity of a mitochondrial DNA gene, the compositioncontaining a probe, a quencher dye, and a fluorescent dye. It is anotherobject of this invention that the quencher dye and fluorescent dye arelinked to the probe. It is a further object of this invention that theprobe has a nucleotide sequence of between approximately any 15contiguous bases and approximately any 45 contiguous bases selected fromthe sequences in SEQ ID NO: 14, 15, 16, and 17, and the reversecomplement thereof.

It is an object of this invention to have a kit or composition that isuseful for determining the integrity of a mitochondrial DNA gene, thekit or composition containing a first polynucleotide and a secondpolynucleotide and, optionally, instructions for using the firstpolynucleotide and second polynucleotide, and optionally, a DNApolymerase. It is another object of this invention that the firstpolynucleotide and second polynucleotide have sequences which are atleast 95% identical to a portion of a mitochondrial DNA gene. It isanother object of this invention that the sequences of the firstpolynucleotide and second polynucleotide are at least 95% identical to aportion of the atp1. It is a further object of this invention that thefirst polynucleotide and the second polynucleotide have the sequences asfollows: the first polynucleotide has SEQ ID NO: 1 and the secondpolynucleotide has SEQ ID NO: 2; the first polynucleotide has SEQ ID NO:3 and the second polynucleotide has SEQ ID NO: 4; the firstpolynucleotide has SEQ ID NO: 5 and the second polynucleotide has SEQ IDNO: 6; and the first polynucleotide has SEQ ID NO: 7 and the secondpolynucleotide has SEQ ID NO: 8. It is a further object of thisinvention that the kit or composition optionally contain a fluorescentcomposition that can be either an intercalating dye or a composition ofa fluorescent dye, a quencher dye and a probe such that the fluorescentdye and quencher dye are linked to the probe. It is another object ofthis invention that the probe have a sequence of between approximatelyfifteen contiguous bases and approximately forty-five contiguous basesof SEQ ID NO: 14 or the reverse complement thereof when the firstpolynucleotide has SEQ ID NO: 1 and the second polynucleotide has SEQ IDNO: 2; or a sequence of between approximately fifteen contiguous basesand approximately forty-five contiguous bases SEQ ID NO: 15 or thereverse complement thereof when the first polynucleotide has SEQ ID NO:3 and the second polynucleotide has SEQ ID NO: 4; or a sequence ofbetween approximately fifteen contiguous bases and approximatelyforty-five contiguous bases of SEQ ID NO: 16 or the reverse complementthereof when the first polynucleotide has SEQ ID NO: 5 and the secondpolynucleotide has SEQ ID NO: 6; or a sequence of between approximatelyfifteen contiguous bases and approximately forty-five contiguous basesof SEQ ID NO: 17 or the reverse complement thereof when the firstpolynucleotide has SEQ ID NO: 7 and the second polynucleotide has SEQ IDNO: 8.

It is an object of this invention to have a method for determining theamount of inactivation of hazardous biological material in a foodmatrix, the method having the steps of exposing the food matrix'sintrinsic DNA to a first polynucleotide, a second polynucleotide, andoptionally a fluorescent composition; amplifying the intrinsic DNA usinga DNA amplification method to produce an amplicon; determining thethreshold cycle of the amplified intrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value of foodmaterial intrinsic DNA that is equivalent to the desired amount ofinactivation of said hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of the firstpolynucleotide at the amplicon's 5′ end and the sequence of the secondpolynucleotide at the amplicon's 3′ end. It is another object of theinvention that the food matrix is plant material, animal material, or acombination thereof. It is another object of this invention that thefluorescent composition can be an intercalating dye or a composition ofa fluorescent dye, a quencher dye and a probe such that the fluorescentdye and quencher dye are linked to the probe and such that the probe hasa sequence of between approximately 15 contiguous bases andapproximately 45 contiguous bases of the amplicon or the reversecomplement thereof.

It is an object of this invention to have a method for determining theamount of inactivation of hazardous biological material in a foodmatrix, the method having the steps of exposing the food matrix'sintrinsic DNA to a first polynucleotide, a second polynucleotide, andoptionally a fluorescent composition; amplifying the intrinsic DNA usinga DNA amplification method to produce an amplicon; determining thethreshold cycle of the amplified intrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value of foodmaterial intrinsic DNA that is equivalent to the desired amount ofinactivation of said hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of the firstpolynucleotide at the amplicon's 5′ end and the sequence of the secondpolynucleotide at the amplicon's 3′ end. It is another object of thisinvention that the first polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof SEQ ID NO: 9, and the second polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof the reverse complement of SEQ ID NO: 9, and the amplicon generated isbetween approximately 80 bp and 250 bp long, is between approximately100 bp and approximately 200 bp long, or is between approximately 125 bpand approximately 175 bp long. It is another object of this inventionthat the fluorescent composition be an intercalating dye or acomposition containing a fluorescent dye, a quencher dye and a probesuch that the fluorescent dye and quencher dye are linked to the probeand such that the probe has a sequence of between approximately 15contiguous bases and approximately 45 contiguous bases of SEQ ID NO: 9or the reverse complement thereof. It is another object of thisinvention to have the step of isolating the intrinsic DNA from the foodmatrix prior to exposing the intrinsic DNA to the first polynucleotideand second polynucleotide. It is another object of the invention thatthe food matrix is plant material, animal material, or a combinationthereof.

It is an object of this invention to have a method for determining theamount of inactivation of hazardous biological material in a foodmatrix, the method having the steps of exposing the food matrix'sintrinsic DNA to a first polynucleotide, a second polynucleotide, andoptionally to a fluorescent composition; amplifying the intrinsic DNAusing a DNA amplification method to produce an amplicon; determining thethreshold cycle of the amplified intrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value of foodmaterial intrinsic DNA that is equivalent to the desired amount ofinactivation of said hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:14. It is another object of this invention that the first polynucleotidehas the sequence of SEQ ID NO: 1, and the second polynucleotide has asequence of SEQ ID NO: 2 or the reverse complement thereof. It isanother object of this invention to have the step of isolating theintrinsic DNA from the food matrix prior to exposing the intrinsic DNAto the first polynucleotide and second polynucleotide. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 14 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theamount of inactivation of hazardous biological material in a foodmatrix, the method having the steps of exposing the food matrix'sintrinsic DNA to a first polynucleotide, a second polynucleotide, andoptionally a fluorescent composition; amplifying the intrinsic DNA usinga DNA amplification method to produce an amplicon; determining thethreshold cycle of the amplified intrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value of foodmaterial intrinsic DNA that is equivalent to the desired amount ofinactivation of said hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:15. It is another object of this invention that the first polynucleotidehas the sequence of SEQ ID NO: 3, and the second polynucleotide has asequence of SEQ ID NO: 4 or the reverse complement thereof. It isanother object of this invention to have the step of isolating theintrinsic DNA from the food matrix prior to exposing the intrinsic DNAto the first polynucleotide and second polynucleotide. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 15 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theamount of inactivation of hazardous biological material in a foodmatrix, the method having the steps of exposing the food matrix'sintrinsic DNA to a first polynucleotide, a second polynucleotide, andoptionally a fluorescent composition; amplifying the intrinsic DNA usinga DNA amplification method to produce an amplicon; determining thethreshold cycle of the amplified intrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value of foodmaterial intrinsic DNA that is equivalent to the desired amount ofinactivation of said hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:16. It is another object of this invention that the first polynucleotidehas the sequence of SEQ ID NO: 5, and the second polynucleotide has asequence of SEQ ID NO: 6 or the reverse complement thereof. It isanother object of this invention to have the step of isolating theintrinsic DNA from the food matrix prior to exposing the intrinsic DNAto the first polynucleotide and second polynucleotide. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 16 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theamount of inactivation of hazardous biological material in a foodmatrix, the method having the steps of exposing the food matrix'sintrinsic DNA to a first polynucleotide, a second polynucleotide, andoptionally a fluorescent composition; amplifying the intrinsic DNA usinga DNA amplification method to produce an amplicon; determining thethreshold cycle of the amplified intrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value of foodmaterial intrinsic DNA that is equivalent to the desired amount ofinactivation of said hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:17. It is another object of this invention that the first polynucleotidehas the sequence of SEQ ID NO: 7, and the second polynucleotide has asequence of SEQ ID NO: 8 or the reverse complement thereof. It isanother object of this invention to have the step of isolating theintrinsic DNA from the food matrix prior to exposing the intrinsic DNAto the first polynucleotide and second polynucleotide. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 17 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theefficacy of a protocol to inactivate hazardous biological material in afood matrix having the steps of, optionally processing a food matrixsample according to the inactivation protocol; exposing the intrinsicDNA of the food matrix processed according to the inactivation protocolto a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA using anamplification process to produce an amplicon; determining the thresholdcycle of the amplified intrinsic DNA; and comparing the obtainedthreshold cycle value to a known threshold cycle value for the intrinsicDNA of the food material that has been processed according to a secondinactivation method known to achieve the desired amount of inactivationof the hazardous biological material. It is a further object of thisinvention that the DNA amplification method use DNA polymerase togenerate an amplicon that has the sequence of the first polynucleotideat the amplicon's 5′ end and the sequence of the second polynucleotideat the amplicon's 3′ end. It is another object of the invention that thefood matrix is plant material, animal material, or a combinationthereof. It is another object of this invention that the firstpolynucleotide and second polynucleotide bind to mtDNA. It is anotherobject of this invention that the first polynucleotide and secondpolynucleotide bind to atp1. It is another object of this invention tooptionally have the step of isolating the intrinsic DNA from theprocessed food matrix prior to exposing the intrinsic DNA to the firstpolynucleotide and second polynucleotide. It is a further object of thisinvention that the fluorescent composition be an intercalating dye or acomposition containing a fluorescent dye, a quencher dye and a probesuch that the fluorescent dye and quencher dye are linked to the probeand such that the probe has a sequence of between approximately 15contiguous bases and approximately 45 contiguous bases of the ampliconor the reverse complement thereof.

It is an object of this invention to have a method for determining theefficacy of a protocol to inactivate hazardous biological material in afood matrix having the steps of, optionally processing a food matrixsample according to the inactivation protocol; exposing the intrinsicDNA of the food matrix processed according to the inactivation protocolto a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA using anamplification process to produce an amplicon; determining the thresholdcycle of the amplified intrinsic DNA; and comparing the obtainedthreshold cycle value to a known threshold cycle value for the intrinsicDNA of the food material that has been processed according to a secondinactivation method known to achieve the desired amount of inactivationof the hazardous biological material. It is another object of thisinvention that the first polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof SEQ ID NO: 9, and the second polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof the reverse complement of SEQ ID NO: 9, and the amplicon generated isbetween approximately 80 bp and 250 bp long, is between approximately100 bp and approximately 200 bp long, or is between approximately 125 bpand approximately 175 bp long. It is a further object of this inventionthat the DNA amplification method use DNA polymerase to generate theamplicon. It is another object of this invention to optionally have thestep of isolating the intrinsic DNA from the processed food matrix priorto exposing the intrinsic DNA to the first polynucleotide and secondpolynucleotide. It is a further object of this invention that thefluorescent composition be an intercalating dye or a compositioncontaining a fluorescent dye, a quencher dye and a probe such that thefluorescent dye and quencher dye are linked to the probe and such thatthe probe has a sequence of between approximately 15 contiguous basesand approximately 45 contiguous bases of the amplicon or the reversecomplement thereof. It is another object of the invention that the foodmatrix is plant material, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theefficacy of a protocol to inactivate hazardous biological material in afood matrix having the steps of, optionally processing a food matrixsample according to the inactivation protocol; optionally isolating theintrinsic DNA of the processed food matrix; exposing the intrinsic DNAof the food matrix processed according to the inactivation protocol to afirst polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA using a DNAamplification method to produce an amplicon; determining the thresholdcycle of the amplified intrinsic DNA; and comparing the obtainedthreshold cycle value to a known threshold cycle value for the intrinsicDNA of the food material that has been processed according to a secondinactivation method known to achieve the desired amount of inactivationof the hazardous biological material. It is a further object of thisinvention that the DNA amplification method use DNA polymerase togenerate an amplicon that has the sequence of SEQ ID NO: 14. It isanother object of this invention that the first polynucleotide has thesequence of SEQ ID NO: 1, and the second polynucleotide has a sequenceof SEQ ID NO: 2 or the reverse complement thereof. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 14 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theefficacy of a protocol to inactivate hazardous biological material in afood matrix having the steps of, optionally processing a food matrixsample according to the inactivation protocol; optionally isolating theintrinsic DNA of the processed food matrix; exposing the intrinsic DNAof the food matrix processed according to the inactivation protocol to afirst polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA using a DNAamplification method to produce an amplicon; determining the thresholdcycle of the amplified intrinsic DNA; and comparing the obtainedthreshold cycle value to a known threshold cycle value for the intrinsicDNA of the food material that has been processed according to a secondinactivation method known to achieve the desired amount of inactivationof the hazardous biological material. It is a further object of thisinvention that the DNA amplification method use DNA polymerase togenerate an amplicon that has the sequence of SEQ ID NO: 15. It isanother object of this invention that the first polynucleotide has thesequence of SEQ ID NO: 3, and the second polynucleotide has a sequenceof SEQ ID NO: 4 or the reverse complement thereof. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 15 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theefficacy of a protocol to inactivate hazardous biological material in afood matrix having the steps of, optionally processing a food matrixsample according to the inactivation protocol; optionally isolating theintrinsic DNA of the processed food matrix; exposing the intrinsic DNAof the food matrix processed according to the inactivation protocol to afirst polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA using a DNAamplification method to produce an amplicon; determining the thresholdcycle of the amplified intrinsic DNA; and comparing the obtainedthreshold cycle value to a known threshold cycle value for the intrinsicDNA of the food material that has been processed according to a secondinactivation method known to achieve the desired amount of inactivationof the hazardous biological material. It is a further object of thisinvention that the DNA amplification method use DNA polymerase togenerate an amplicon that has the sequence of SEQ ID NO: 16. It isanother object of this invention that the first polynucleotide has thesequence of SEQ ID NO: 5, and the second polynucleotide has a sequenceof SEQ ID NO: 6 or the reverse complement thereof. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 16 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for determining theefficacy of a protocol to inactivate hazardous biological material in afood matrix having the steps of, optionally processing a food matrixsample according to the inactivation protocol; optionally isolating theintrinsic DNA of the processed food matrix; exposing the intrinsic DNAof the food matrix processed according to the inactivation protocol to afirst polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA using a DNAamplification method to produce an amplicon; determining the thresholdcycle of the amplified intrinsic DNA; and comparing the obtainedthreshold cycle value to a known threshold cycle value for the intrinsicDNA of the food material that has been processed according to a secondinactivation method known to achieve the desired amount of inactivationof the hazardous biological material. It is a further object of thisinvention that the DNA amplification method use DNA polymerase togenerate an amplicon that has the sequence of SEQ ID NO: 17. It isanother object of this invention that the first polynucleotide has thesequence of SEQ ID NO: 7, and the second polynucleotide has a sequenceof SEQ ID NO: 8 or the reverse complement thereof. It is a furtherobject of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 17 or the reverse complement thereof. Itis another object of the invention that the food matrix is plantmaterial, animal material, or a combination thereof.

It is an object of this invention to have a method for assessing theefficacy of a protocol for inactivation of hazardous material in or onan item, the method having the steps of optionally processing a sampleof extrinsic DNA according to the protocol; optionally isolating theprocessed extrinsic DNA; exposing the extrinsic DNA to a firstpolynucleotide, to a second polynucleotide, and optionally a fluorescentcomposition; amplifying the extrinsic DNA using a DNA amplificationmethod to produce an amplicon; determining the threshold cycle of theextrinsic DNA; and comparing the obtained threshold cycle value to aknown threshold cycle value for extrinsic DNA that has been processedaccording to a second inactivation method known to achieve the desiredamount of inactivation of the hazardous biological material. It is afurther object of this invention that the DNA amplification method useDNA polymerase to generate an amplicon that has the sequence of thefirst polynucleotide at the amplicon's 5′ end and the sequence of thesecond polynucleotide at the amplicon's 3′ end. It is a further objectof this invention that the fluorescent composition be an intercalatingdye or a composition containing a fluorescent dye, a quencher dye and aprobe such that the fluorescent dye and quencher dye are linked to theprobe and such that the probe has a sequence of between approximately 15contiguous bases and approximately 45 contiguous bases of the ampliconor the reverse complement thereof. It is a further object of thisinvention that the item be a food matrix, a container, equipment, or amedical device.

It is an object of this invention to have a method for assessing theefficacy of a protocol for inactivation of hazardous material in or onan item, the method having the steps of optionally processing a sampleof extrinsic DNA according to the protocol; optionally isolating theprocessed extrinsic DNA; exposing the extrinsic DNA to a firstpolynucleotide, to a second polynucleotide, and optionally to afluorescent composition; amplifying the extrinsic DNA using a DNAamplification method to produce an amplicon; determining the thresholdcycle of the extrinsic DNA; and comparing the obtained threshold cyclevalue to a known threshold cycle value for extrinsic DNA that has beenprocessed according to a second inactivation method known to achieve thedesired amount of inactivation of the hazardous biological material. Itis a further object of this invention that the DNA amplification methoduse DNA polymerase to generate an amplicon that has the sequence of thefirst polynucleotide at the amplicon's 5′ end and the sequence of thesecond polynucleotide at the amplicon's 3′ end. It is another object ofthis invention that the first polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof any mtDNA gene, and the second polynucleotide has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of the reverse complement of the same mtDNA gene, andthe amplicon generated is between approximately 80 bp and 250 bp long,is between approximately 100 bp and approximately 200 bp long, or isbetween approximately 125 bp and approximately 175 bp long. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of the amplicon or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a method for assessing theefficacy of a protocol for inactivation of hazardous material in or onan item, the method having the steps of optionally processing a sampleof extrinsic DNA according to the protocol; optionally isolating theprocessed extrinsic DNA; exposing the extrinsic DNA to a firstpolynucleotide, a second polynucleotide, and optionally a fluorescentcomposition; amplifying the extrinsic DNA using a DNA amplificationmethod to produce an amplicon; determining the threshold cycle of theextrinsic DNA; and comparing the obtained threshold cycle value to aknown threshold cycle value for extrinsic DNA that has been processedaccording to a second inactivation method known to achieve the desiredamount of inactivation of the hazardous biological material. It is afurther object of this invention that the DNA amplification method useDNA polymerase to generate an amplicon that has the sequence of thefirst polynucleotide at the amplicon's 5′ end and the sequence of thesecond polynucleotide at the amplicon's 3′ end. It is another object ofthis invention that the first polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof SEQ ID NO: 9, and the second polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof the reverse complement of SEQ ID NO: 9, and the amplicon generated isbetween approximately 80 bp and 250 bp long, is between approximately100 bp and approximately 200 bp long, or is between approximately 125 bpand approximately 175 bp long. It is a further object of this inventionthat the fluorescent composition be an intercalating dye or acomposition containing a fluorescent dye, a quencher dye and a probesuch that the fluorescent dye and quencher dye are linked to the probeand such that the probe has a sequence of between approximately 15contiguous bases and approximately 45 contiguous bases of the ampliconor the reverse complement thereof. It is a further object of thisinvention that the item be a food matrix, a container, equipment, or amedical device.

It is an object of this invention to have a method for assessing theefficacy of a protocol for inactivation of hazardous material in or onan item, the method having the steps of optionally processing a sampleof extrinsic DNA according to the protocol; optionally isolating theprocessed extrinsic DNA; exposing the extrinsic DNA to a firstpolynucleotide, a second polynucleotide, and optionally a fluorescentcomposition; amplifying the extrinsic DNA using a DNA amplificationmethod to produce an amplicon; determining the threshold cycle of theextrinsic DNA; and comparing the obtained threshold cycle value to aknown threshold cycle value for extrinsic DNA that has been processedaccording to a second inactivation method known to achieve the desiredamount of inactivation of the hazardous biological material. It is afurther object of this invention that the DNA amplification method useDNA polymerase to generate an amplicon that has the sequence of SEQ IDNO: 14; that the first polynucleotide has the sequence of SEQ ID NO: 1,and the second polynucleotide has the sequence of SEQ ID NO: 2. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 14 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a method for assessing theefficacy of a protocol for inactivation of hazardous material in or onan item, the method having the steps of optionally processing a sampleof extrinsic DNA according to the protocol; optionally isolating theprocessed extrinsic DNA; exposing the extrinsic DNA to a firstpolynucleotide, a second polynucleotide, and optionally a fluorescentcomposition; amplifying the extrinsic DNA using a DNA amplificationmethod to produce an amplicon; determining the threshold cycle of theextrinsic DNA; and comparing the obtained threshold cycle value to aknown threshold cycle value for extrinsic DNA that has been processedaccording to a second inactivation method known to achieve the desiredamount of inactivation of the hazardous biological material. It is afurther object of this invention that the DNA amplification method useDNA polymerase to generate an amplicon that has the sequence of SEQ IDNO: 15; that the first polynucleotide has the sequence of SEQ ID NO: 3,and the second polynucleotide has the sequence of SEQ ID NO: 4. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 15 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a method for assessing theefficacy of a protocol for inactivation of hazardous material in or onan item, the method having the steps of optionally processing a sampleof extrinsic DNA according to the protocol; optionally isolating theprocessed extrinsic DNA; exposing the extrinsic DNA to a firstpolynucleotide, a second polynucleotide, and optionally a fluorescentcomposition; amplifying the extrinsic DNA using a DNA amplificationmethod to produce an amplicon; determining the threshold cycle of theextrinsic DNA; and comparing the obtained threshold cycle value to aknown threshold cycle value for extrinsic DNA that has been processedaccording to a second inactivation method known to achieve the desiredamount of inactivation of the hazardous biological material. It is afurther object of this invention that the DNA amplification method useDNA polymerase to generate an amplicon that has the sequence of SEQ IDNO: 16; that the first polynucleotide has the sequence of SEQ ID NO: 5,and the second polynucleotide has the sequence of SEQ ID NO: 6. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 16 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a method for assessing theefficacy of a protocol for inactivation of hazardous material in or onan item, the method having the steps of optionally processing a sampleof extrinsic DNA according to the protocol; optionally isolating theprocessed extrinsic DNA; exposing the extrinsic DNA to a firstpolynucleotide, a second polynucleotide, and optionally a fluorescentcomposition; amplifying the extrinsic DNA using a DNA amplificationmethod to produce an amplicon; determining the threshold cycle of theextrinsic DNA; and comparing the obtained threshold cycle value to aknown threshold cycle value for extrinsic DNA that has been processedaccording to a second inactivation method known to achieve the desiredamount of inactivation of the hazardous biological material. It is afurther object of this invention that the DNA amplification method useDNA polymerase to generate an amplicon that has the sequence of SEQ IDNO: 17; that the first polynucleotide has the sequence of SEQ ID NO: 7,and the second polynucleotide has the sequence of SEQ ID NO: 8. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 17 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a quality control method fordetermining the amount of inactivation of a hazardous material in or onan item, the method having the steps of optionally processing a samplecontaining either intrinsic DNA or extrinsic DNA according to thepre-determined inactivation protocol; optionally isolating the processedintrinsic DNA or extrinsic DNA; exposing the intrinsic DNA or extrinsicDNA to a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA or extrinsic DNAusing a DNA amplification method to produce an amplicon; determining thethreshold cycle of the intrinsic DNA or extrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value forintrinsic DNA or extrinsic DNA that is equivalent to the desired amountof inactivation of the hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that is between approximately 80 bpand approximately 250 bp long and that has the sequence of the firstpolynucleotide at the amplicon's 5′ end and the sequence of the secondpolynucleotide at the amplicon's 3′ end. It is a further object of thisinvention that the fluorescent composition be an intercalating dye or acomposition containing a fluorescent dye, a quencher dye and a probesuch that the fluorescent dye and quencher dye are linked to the probeand such that the probe has a sequence of between approximately 15contiguous bases and approximately 45 contiguous bases of the ampliconor the reverse complement thereof. It is a further object of thisinvention that the item be a food matrix, a container, equipment, or amedical device.

It is an object of this invention to have a quality control method fordetermining the amount of inactivation of a hazardous material in or onan item, the method having the steps of optionally processing a samplecontaining either intrinsic DNA or extrinsic DNA according to thepre-determined inactivation protocol; optionally isolating the processedintrinsic DNA or extrinsic DNA; exposing the intrinsic DNA or extrinsicDNA to a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA or extrinsic DNAusing a DNA amplification method to produce an amplicon; determining thethreshold cycle of the intrinsic DNA or extrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value forintrinsic DNA or extrinsic DNA that is equivalent to the desired amountof inactivation of the hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of the firstpolynucleotide at the amplicon's 5′ end and the sequence of the secondpolynucleotide at the amplicon's 3′ end. It is another object of thisinvention that the first polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof SEQ ID NO: 9, and the second polynucleotide has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof the reverse complement of SEQ ID NO: 9, and the amplicon generated isbetween approximately 80 bp and 250 bp long, is between approximately100 bp and approximately 200 bp long, or is between approximately 125 bpand approximately 175 bp long. It is a further object of this inventionthat the fluorescent composition be an intercalating dye or acomposition containing a fluorescent dye, a quencher dye and a probesuch that the fluorescent dye and quencher dye are linked to the probeand such that the probe has a sequence of between approximately 15contiguous bases and approximately 45 contiguous bases of the ampliconor the reverse complement thereof. It is a further object of thisinvention that the item be a food matrix, a container, equipment, or amedical device.

It is an object of this invention to have a quality control method fordetermining the amount of inactivation of a hazardous material in or onan item, the method having the steps of optionally processing a samplecontaining either intrinsic DNA or extrinsic DNA according to thepre-determined inactivation protocol; optionally isolating the processedintrinsic DNA or extrinsic DNA; exposing the intrinsic DNA or extrinsicDNA to a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA or extrinsic DNAusing a DNA amplification method to produce an amplicon; determining thethreshold cycle of the intrinsic DNA or extrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value forintrinsic DNA or extrinsic DNA that is equivalent to the desired amountof inactivation of the hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:14; that the first polynucleotide has the sequence of SEQ ID NO: 1, andthe second polynucleotide has the sequence of SEQ ID NO: 2. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 14 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a quality control method fordetermining the amount of inactivation of a hazardous material in or onan item, the method having the steps of optionally processing a samplecontaining either intrinsic DNA or extrinsic DNA according to thepre-determined inactivation protocol; optionally isolating the processedintrinsic DNA or extrinsic DNA; exposing the intrinsic DNA or extrinsicDNA to a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA or extrinsic DNAusing a DNA amplification method to produce an amplicon; determining thethreshold cycle of the intrinsic DNA or extrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value forintrinsic DNA or extrinsic DNA that is equivalent to the desired amountof inactivation of the hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:15; that the first polynucleotide has the sequence of SEQ ID NO: 3, andthe second polynucleotide has the sequence of SEQ ID NO: 4. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 15 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a quality control method fordetermining the amount of inactivation of a hazardous material in or onan item, the method having the steps of optionally processing a samplecontaining either intrinsic DNA or extrinsic DNA according to thepre-determined inactivation protocol; optionally isolating the processedintrinsic DNA or extrinsic DNA; exposing the intrinsic DNA or extrinsicDNA to a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA or extrinsic DNAusing a DNA amplification method to produce an amplicon; determining thethreshold cycle of the intrinsic DNA or extrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value forintrinsic DNA or extrinsic DNA that is equivalent to the desired amountof inactivation of the hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:16; that the first polynucleotide has the sequence of SEQ ID NO: 5, andthe second polynucleotide has the sequence of SEQ ID NO: 6. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 16 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a quality control method fordetermining the amount of inactivation of a hazardous material in or onan item, the method having the steps of optionally processing a samplecontaining either intrinsic DNA or extrinsic DNA according to thepre-determined inactivation protocol; optionally isolating the processedintrinsic DNA or extrinsic DNA; exposing the intrinsic DNA or extrinsicDNA to a first polynucleotide, a second polynucleotide, and optionally afluorescent composition; amplifying the intrinsic DNA or extrinsic DNAusing a DNA amplification method to produce an amplicon; determining thethreshold cycle of the intrinsic DNA or extrinsic DNA; and comparing theobtained threshold cycle value to a known threshold cycle value forintrinsic DNA or extrinsic DNA that is equivalent to the desired amountof inactivation of the hazardous biological material. It is a furtherobject of this invention that the DNA amplification method use DNApolymerase to generate an amplicon that has the sequence of SEQ ID NO:17; that the first polynucleotide has the sequence of SEQ ID NO: 7, andthe second polynucleotide has the sequence of SEQ ID NO: 8. It is afurther object of this invention that the fluorescent composition be anintercalating dye or a composition containing a fluorescent dye, aquencher dye and a probe such that the fluorescent dye and quencher dyeare linked to the probe and such that the probe has a sequence ofbetween approximately 15 contiguous bases and approximately 45contiguous bases of SEQ ID NO: 17 or the reverse complement thereof. Itis a further object of this invention that the item be a food matrix, acontainer, equipment, or a medical device.

It is an object of this invention to have a quality control method fordetermining the amount of inactivation of a hazardous material in or onan item, the method having the steps of optionally processing a samplecontaining either intrinsic DNA or extrinsic DNA according to thepre-determined inactivation protocol; optionally isolating the processedintrinsic DNA or extrinsic DNA; running the intrinsic DNA or extrinsicDNA on a electrophoretic gel; determining the amount of DNAfragmentation for DNA sizes ranging from approximately 35 bp toapproximately 10,380 bp; determining the DNA integrity number; andcomparing the obtained DNA integrity number to a known DNA integritynumber that is equivalent to the desired amount of inactivation of thehazardous biological material. It is a further object of this inventionthat the intrinsic DNA or extrinsic DNA be exposed to a fluorescentcomposition prior to or after running the intrinsic DNA or extrinsic DNAon the electrophoretic gel; such that the fluorescent composition is afluorescent dye for imaging DNA. It is a further object of thisinvention that the item be a food matrix, a container, equipment, or amedical device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the standard curve for qPCR for mtDNA copy number.The standard curve is also used to determine amplification efficiency ofthe protocol (106%), the limit of detection (10 copies) and the lineardynamic range (7 orders of magnitude).

FIG. 2 illustrates the effect of hot oil bath (at 121° C.) on sweetpotato puree mtDNA fragmentation (increase in Ct value) over time.

FIG. 3 illustrates the high correlation between surrogate GS sporedestruction and increase in Ct value of sweet potato puree mtDNA overtime for the hot oil assay (121° C.).

FIG. 4 illustrates the high correlation (R²=0.87) between surrogate G.stearothermophilus spore destruction and increase in Ct value over timewhen the sweet potato puree and G. stearothermophilus spore areautoclaved (121° C.).

FIG. 5 illustrates the change in Ct value indicating mtDNA fragmentationof cucumbers during pasteurization (75° C. for 15 minutes), fermentationand storage compared to Ct values of cucumbers in autoclave treatment(121° C.).

FIG. 6 illustrates the mtDNA degradation (increase in Ct value) ofpeanuts dry roasted (167° C.) at the indicated time intervals.

FIG. 7 illustrates the correlation between mtDNA degradation (increasein Ct value) in dry roast treatment of peanuts (167° C.) and survival ofa Salmonella surrogate, E. faecium (log₁₀ CFU/ml).

FIG. 8 compares mtDNA degradation (increase in Ct value) of dry roastedpeanuts (167° C.) with the Hunter L color, a roasting quality indicator.

FIG. 9 shows the sequence of I. batatas atp1, the location of theprimers used, and the sequences of the amplicons generated.

FIG. 10 shows the extrinsic DNA thermometer at three different pH levelsfrom 0-24 minutes at 96° C.

FIG. 11 shows the extrinsic DNA thermometer at three different pH levelsfrom 0-5 minutes at 96° C.

FIG. 12 illustrates the fragmentation and reduction of total DNAintegrity caused by autoclave treatment by comparing size andconcentrations of cucumber DNA globally.

FIG. 13 is a flow chart illustrating the invention described herein.

FIG. 14A illustrates the correlation between Ct values of sweet potatomtDNA and F values of inactivation of G. stearothermophilus in cannedsweet potato using substandard 12-D protocol (protocol 00). FIG. 14Billustrates the correlation between Ct values of sweet potato mtDNA andF values of inactivation of G. stearothermophilus in canned sweet potatousing standard 12-D protocol (protocol 01).

FIG. 15 illustrates the slopes used to calculate D values for timed oilbath treatments for G. stearothermophilus spores at 116° C., 121° C.,123° C., and 126° C. for the indicated times.

FIG. 16 illustrates the slope used to calculate the z-value for timedoil bath treatments for G. stearothermophilus spores at 116° C., 121°C., 123° C., and 126° C. for the indicated times.

FIG. 17 illustrates sweet potato puree mtDNA fragmentation and D valuesin hot oil bath at 116° C., 121° C., 123° C., and 126° C. for theindicated times.

FIG. 18 illustrates the slope used to calculate z-value for sweet potatopuree mtDNA fragmentation in hot oil bath at 116° C., 121° C., 123° C.,and 126° C. for the indicated times.

FIG. 19 illustrates the linear relationship between sweet potato pureemtDNA fragmentation in hot oil bath at 121° C. versus G.stearothermophilus spore counts in hot oil bath at 121° C.

DETAILED DESCRIPTION OF THE INVENTION

This invention determines inactivation of biological material, primarilyhazardous biological material such as in or on food or an item byquantifying the amount of degradation in a food matrix's intrinsic DNAor in extrinsic DNA added to food or an item. The amount of DNAdegradation is correlated to inactivation of hazardous biologicalmaterial such as disease causing pathogens, and/or widely acceptedsurrogates for bacterial pathogen's spores. More specifically, thequantified DNA degradation is a time/temperature indicator which is asurrogate for inactivation of hazardous biological material. When DNAdegradation is at or after specified values, the inactivation ofhazardous biological material in or on a food or item is assured.Because this process uses quantitative PCR, some commercially availablereagents, and/or apparatuses, the assay is inexpensive and simple touse. Further, it provides an answer regarding inactivation of thehazardous biological material of interest significantly faster thanprior art methods of culturing for biological material, and moreaccurately over prior art methods of using PCR to detect the presence ofa pathogen's DNA. One method for determining DNA degradation is byperforming quantitative PCR (qPCR) on specific genes contained inmitochondrial DNA (mtDNA) of the food matrix. A second method is toexamine DNA integrity of a food matrix. A third method is to add mtDNA(extrinsic DNA) as an extrinsic source in a recoverable container. Allthree methods are used as time/temperature integrators. These assaysserve as presumptive verification of processing efficacy. Using the toolbox approach, these assays provide rapid results that a processor canuse to evaluate the reduction in amount of viable hazardous biologicalmaterial in or on an item (food, device, etc.) prior to shipping orusing the item. These assays can also be used to evaluate deviations innormal processing of items and to evaluate the efficacy of newprocessing methods under consideration. These assays use mitochondrialDNA (mtDNA) fragmentation or DNA integrity to determine the degradationof DNA over the range of time, temperature, and other inactivationprocess' conditions such as, but not limited to, high pressure orultraviolet light. MtDNA is a surrogate for the inactivation ofhazardous biological material in or on the item being treated (food,device, etc.). DNA integrity, whether measuring intrinsic DNA integrityor extrinsic DNA integrity, is a surrogate for the inactivation ofhazardous biological material in or on the item being treated (food,device, etc.). These methods can be used to determine if an appropriatereduction in viable hazardous biological material (bacteria, bacterialspores, viruses, fungi, parasites, cancer cells, etc.) occurs after theprocessing of the food matrix or device. An appropriate reduction inviable hazardous biological material could be a 5 log reduction of aparticular pathogen or any other amount desired or required byregulations governing food safety or medical device safety. Thesemethods can also be used as quality control for a particularinactivation process. These methods can also be used to assay thesurvival of hazardous biological material after processing.

Polymerase chain reaction (or PCR) is a technique to copy (or amplify) asmall quantity of DNA. Using PCR, one can generate greater than100,000,000 or even one billion copies of the desired DNA within acouple of hours. To amplify a segment of DNA using PCR, the sample isfirst heated so the DNA denatures (separates into two pieces ofsingle-stranded DNA). Next, the sample is cooled to a temperature lowerthan the melting (or denaturing) temperature of the DNA but stillsubstantially higher than room temperature. At this temperature primersbind specific, pre-determined sites. Taq polymerase (a DNA polymeraseactive at high temperatures) synthesizes two new strands of DNA, usingthe original strands as templates and primers that bind to the originalstrands of DNA as initiation points for DNA extension by Taq polymerase.Of course, sufficient amounts of free nucleic acids are added to thereaction mixture for use by Taq polymerase to generate the new DNA. Thisprocess results in the duplication of a section of the original DNAbased on the binding location of the primers. Each new DNA segment (alsoreferred to as an amplicon) contains one old and one new strand of DNA.The sample is heated again to denature the DNA again and allowed to coolso that Taq polymerase can generate new amplicons. The cycle ofdenaturing and synthesizing new DNA is repeated as many as thirty orforty times, leading to more than one billion amplicons. A thermocycleris a programmable apparatus that automates the temperature changesutilized in PCR, controlling DNA denaturation and synthesis. PCR can becompleted in a few hours. Some early U.S. patents on PCR include U.S.Pat. Nos. 4,683,195; 4,683,202; and 4,800,159.

Quantitative PCR (qPCR), also called real-time PCR, involves monitoringDNA amplification during each cycle of PCR using fluorescent label. Whenthe DNA is in the log linear phase of amplification, the amount offluorescence increases above the background. The point at which thefluorescence becomes measurable is called the Threshold Cycle (Ct) orcrossing point. By using multiple dilutions of a known amount ofstandard DNA, a standard curve can be generated of log concentrationagainst Ct (see FIG. 1). The amount of DNA or cDNA in an unknown samplecan then be calculated from its Ct value. Two types of fluorescentlabels are used with qPCR. One label is an intercalating dye thatincorporates into double-stranded DNA, such as, but not limited to,SYBR® Green. An intercalating dye is appropriate when a single ampliconis being studied. The second type of fluorescent label is a probe thatbinds specifically to the target DNA, such as TaqMan® probes, MolecularBeacons™, or Scorpion primers. The probe is labeled with a fluorescentdye (such as, but not limited to Texas Red®, FAM, TET, HEX, TAMRA, JOE,and ROX) and a quencher (such as, but not limited to Dabcyl and Dabsyl).The oligonucleotide itself has no significant fluorescence, butfluoresces either when annealed to the template (as in MolecularBeacons™) or when the dye is clipped from the oligonucleotide duringextension (as in TaqMan® probes). Multiplex PCR is possible by usingdyes with different fluorescent emissions for each probe. Thefluorescent compositions described herein are simply examples ofcompositions for imaging, identifying, and/or quantifying DNA. Insteadof the fluorescent compositions described herein, one can label DNA withcompositions that are known in the art (some of which are describedinfra) or that are developed in the future. These labels can be used toimage, identify, and/or quantify DNA using similar methods as describedherein. The fluorescent compositions are simply one well-known andwell-accepted compositions for imaging, identifying, and/or quantifyingDNA for the methods described herein.

The term “nucleic acid” as used herein, refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid”polymers occur in either single- or double-stranded form, but are alsoknown to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers aswell as nucleic acids comprising known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Exemplary analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs) and locked nucleic acids (RNA monomers with a modifiedbackbone). “DNA”, “RNA”, “polynucleotides”, “polynucleotide sequence”,“oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acidmolecule”, “nucleic acid sequence”, “nucleic acid fragment”, and“isolated nucleic acid fragment” are used interchangeably herein.

The term “label” as used herein, refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. Exemplary labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available.

As used herein a nucleic acid “probe”, oligonucleotide “probe”, orsimply a “probe” refers to a nucleic acid capable of binding to a targetnucleic acid of complementary sequence through one or more types ofchemical bonds, usually through complementary base pairing, usuallythrough hydrogen bond formation. As used herein, a probe may includenatural (i.e., A, G, C, or T) or modified bases (e.g., 7-deazaguanosine,inosine, etc.). In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. In one exemplary embodiment, probes aredirectly labeled as with isotopes, chromophores, lumiphores, chromogensetc. In other exemplary embodiments probes are indirectly labeled e.g.,with biotin to which a streptavidin complex may later bind. By assayingfor the presence or absence of the probe, one can detect the presence orabsence of the select sequence or subsequence.

Thus, the term “probe” as used herein refers to a probe that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

The term “primer” as used herein, refers to short nucleic acids,typically a DNA oligonucleotide of at least about 15 nucleotides inlength. In an exemplary embodiment, primers are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand. Annealed primersare then extended along the target DNA strand by a DNA polymeraseenzyme. Primer pairs can be used for amplification of a nucleic acidsequence, e.g., by the polymerase chain reaction (PCR) or othernucleic-acid amplification methods known in the art.

PCR primer pairs are typically derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5 ©1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). One of ordinary skill in the art will appreciate thatthe specificity of a particular probe or primer increases with itslength. Thus, for example, a primer comprising 20 consecutivenucleotides of a promoter complex sequence will anneal to a relatedtarget sequence with a higher specificity than a corresponding primer ofonly 15 nucleotides. Thus, in an exemplary embodiment, greaterspecificity of a nucleic acid primer or probe is attained with probesand primers selected to comprise 20, 25, 30, 35, 40, 50 or moreconsecutive nucleotides of a selected sequence. When discussing primerpairs, one provides the sequence of both the forward and reverse primersin the 5′ to 3′ direction and is the sequence of the positive strand ofDNA. However, when performing PCR, the sequence of the reverse primeractually used is the reverse complement of the sequence of the reverseprimer. Thus for 174R primer, the actual sequence used is in SEQ ID NO:10; for 108R primer, the actual sequence uses is in SEQ ID NO: 11; for81R primer, the actual sequence used is in SEQ ID NO: 12; and for 141Rprimer, the actual sequence used is in SEQ ID NO: 13.

Nucleic acid probes and primers are readily prepared based on thenucleic acid sequences disclosed herein. Methods for preparing and usingprobes and primers and for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g., in Sambrook etal., Molecular Cloning, A Laboratory Manual 2nd ed. 1989, Cold SpringHarbor Laboratory; and Current Protocols in Molecular Biology, Ausubelet al., eds., 1994, John Wiley & Sons).

The term “capable of hybridizing under stringent hybridizationconditions” as used herein, refers to annealing a first nucleic acid toa second nucleic acid under stringent hybridization conditions (definedbelow). In an exemplary embodiment, the first nucleic acid is a testsample, and the second nucleic acid is the sense or antisense strand ofa nucleic acid of interest. Hybridization of the first and secondnucleic acids is conducted under standard stringent conditions, e.g.,high temperature and/or low salt content, which tend to disfavorhybridization of dissimilar nucleotide sequences.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., 85% identity, 90% identity, 99%, or 100% identity), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using a sequence comparison algorithm orby manual alignment and visual inspection.

The phrase “substantially identical”, in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least about 85%, identity, at least about 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. In an exemplary embodiment, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length. In another exemplary embodiment, thesubstantial identity exists over a region of the sequences that is atleast about 100 residues in length. In still another exemplaryembodiment, the substantial identity exists over a region of thesequences that is at least about 150 residues or more, in length. In oneexemplary embodiment, the sequences are substantially identical over theentire length of nucleic acid or protein sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from about 20 to about 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

An exemplary algorithm for sequence comparison is PILEUP. PILEUP createsa multiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., 1984. Nuc. Acids Res. 12:387-395.

The phrase “selectively hybridizes to” or “specifically hybridizes to”refers to the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA). In general, two nucleic acid sequences are saidto be “substantially identical” when the two molecules or theircomplements selectively or specifically hybridize to each other understringent conditions.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For high stringency hybridization, a positivesignal is at least two times background, preferably 10 times backgroundhybridization. Exemplary high stringency or stringent hybridizationconditions include: 50% formamide, 5×SSC and 1% SDS incubated at 42° C.or 5×SSC and 1% SDS incubated at 65° C., with a wash in 0.2×SSC and 0.1%SDS at 65° C. However, other high stringency hybridization conditionsknown in the art can be used.

Intrinsic DNA means nucleic acids (DNA or RNA) that are presentnaturally in a sample, including nucleic acids from hazardous biologicalmaterial present in the sample. For example, intrinsic DNA for a foodmatrix is the DNA and RNA in the cells of the plant or animalingredients in the food matrix. Intrinsic DNA includes mitochondrial DNA(mtDNA), but is not limited to mtDNA.

Extrinsic DNA means any nucleic acids (DNA or RNA) added to a system forinactivating hazardous biological material. Usually, the extrinsic DNAis double-stranded polynucleotide which can range from betweenapproximately 80 bp to approximately 250 bp long. Extrinsic DNA does notneed to be from any particular gene or non-coding region. However, onemust have primers, and optionally a labeled probe, which can be usedwhile performing qPCR on the extrinsic DNA. The extrinsic DNA is usedwhen one does not have or does not want to perform qPCR on intrinsic DNAto determine the efficacy of an inactivation process. For example, whendetermining if a certain process is sufficient robust to destroyhazardous biological material present on an item (e.g., a jar or medicaldevice), one subjects a test sample of extrinsic DNA in a low pHsolution or high pH solution to the inactivation process. An item,optionally, may or may not be subjected to the inactivation process withthe extrinsic DNA. Then one performs the qPCR assays described herein onthe extrinsic DNA to determine the amount of nucleic acid degradationand correlate that degradation to the destruction/inactivation ofhazardous biological material on/in the item.

DNA degradation, RNA degradation, nucleic acid degradation are thebreaking of the chemical bonds with the nucleic acids so that theorganism containing those degraded nucleic acids is not viable. Nucleicacid degradation can occur when nucleic acids are exposed to certainconditions, such as, elevated temperature, acidity, alkalinity, salt,preservatives, UV light, high pressure, to name a few.

DNA integrity is the wholeness or completeness of a cell's genomic andorganelle-based DNA. After heat treatments, such as autoclaving, orother types of conditions that degrades a cell's nucleic acids, thecell's DNA integrity is reduced. The amount fragmentation (reduction inintegrity) can be measured globally and is used as a time/temperatureintegrator. One can measure the integrity of either intrinsic DNA orextrinsic DNA to determine the amount of inactivation of the hazardousbiological material.

Hazardous biological material is any biological material that could harman animal if the animal ingests the biological material or, if placed onor inside the animal. Hazardous biological material include, but are notlimited to, toxins, viruses, parasites, fungi, bacteria, spores(bacterial, fungal or parasitical), other types of pathogens, and cancercells.

Mitochondria are “power-house” organelles found in multiple numbers inall cells of eukaryotes, and each mitochondrion possesses its own genomein multiple copies. Mitochondrial DNA contains polynucleotide sequencesthat are species-specific or family-specific. In addition, themitochondrial genome also contains polynucleotide sequences that arehighly conserved in many eukaryotic plants or animals. These propertiesmake mtDNA sequences excellent targets for amplification in terms ofspecificity, sensitivity and robustness in addition to the fact thatmultiple copies per cell (>1,000) exist. Therefore, the advantages oftargeting mtDNA with qPCR are substantial.

All mtDNA qPCR assays for this invention meet MIQE Guidelines (Bustin,et al., Clinical Chem. 55:611-622 (2009)) or Minimum Information forpublication of Quantitative real-time PCR Experiments which features aquality control checklist on sample processing, nucleic acid extraction,target amplicon specifications, reaction optimization, specificity ofreaction, internal amplification controls (IAC), calibration curves withcalculated PCR efficiency, linear dynamic range and data analysisincluding repeatability and statistical methods. This approach tomonitoring food safety and cleaning of objects, in general, represents aparadigm shift by using qPCR to quantify the disappearance of mtDNA overtime caused by thermal or microwave processing and correlating thedegradation rates closely to the thermal death time (D) of spore-formingbacteria. This method also involves correlating the thermal death time(D) of the bacteria, as determined by culture methods, to thedestruction of mtDNA of the thermally- or microwave-processed foodstuff.Assessing mtDNA decomposition over time can also be used to assess shelflife of a food and the level of inactivation of biological material onor in medical equipment, empty or filled food containers, or otheritems.

The mtDNA qPCR assays of this invention are adjusted to highly correlateto D values (time required for 1 log reduction of pathogens at a certaintemperature) according to the length of the amplicon, secondary DNAstructure, annealing temperature, use of locked nucleic acids andprimer/probe efficiencies.

As used herein, the term “about” and “approximately” refers to aquantity, level, value or amount that varies by as much as 30%,preferably by as much as 20%, and more preferably by as much as 10% to areference quantity, level, value or amount. All cited prior artdocuments are incorporated by reference.

Example 1 Primers for Plant mtDNA Selection and Generation ofStandardized Curves

Primers are designed which use consensus sequences to target a widevariety of plant foods. Four sets of qPCR primers shown in Table 1 aredesigned with Primer Quest software(http://scitools.idtdna.com/Primerquest/) from Integrated DNATechnologies, Inc. (IDT) (Coralville, Iowa) targeting the Ipomoeabatatas F1-ATPase alpha subunit (atp1) mitochondrial gene (GenBankAY596672.1). Amplicon length for each primer set is indicated by thenumber in the primer sets' name, 81 bp, 108 bp, 141 bp, and 174 bp. FIG.9 shows the sequence of I. batatas atp1 (SEQ ID NO: 9), the location ofthe primers used, and the sequences of the amplicons generated. Allprimer sets match the Ipomoea batatas atp1 gene with 100% identity, andbetween approximately 95% and 100% identity for a wide range of commonfruits, vegetables, and nuts (see Table 2 infra) when subjected to NCBIBLAST searches. Primers are purchased from IDT (Coralville, Iowa).Oligonucleotide primers are reconstituted in TE buffer (pH 7.5) andstored at −20° C. prior to use.

TABLE 1 Primer Start name Position Sequence 174F  6985′-TTTCCGCGATAATGGAATGCACGC-3′ (forward) (SEQ ID NO: 1) 174R  8715′-TCCGATCGTTTAGCCGCTCTTTCT-3′ (reverse) (SEQ ID NO: 2) 108F 11335′-CGCCTTTGCTCAATTTGGCTCAGA-3′ (forward) (SEQ ID NO: 3) 108R 12405′-GGCAGTGGTGCATATTGTGGTTGT-3′ (reverse) (SEQ ID NO: 4) 81F 11335′-CGCCTTTGCTCAATTTGGCTCAGA-3′ (forward) (SEQ ID NO: 5) 81R 12135′-AGTACTTCTGTCAGCCTTGCACCT-3′ (reverse) (SEQ ID NO: 6) 141F   875′-GAATTTGCCAGCGGTGTGAAAGGA-3′ (forward) (SEQ ID NO: 7) 141R  2275′-TCCCGCAGGAACATCCACAATAGA-3′ (reverse) (SEQ ID NO: 8)

A test comparing autoclaved (steamed at 121° C. for 20 minutes) versusnon-autoclaved sweet potato puree DNA is run with each primer set. Pureeis generated prior to treatment by grinding the sweet potato and waterin Waring blender until puree is formed. Sweet potato DNA is thenisolated after treatment using MO BIO PowerSoil® DNA isolation kit(Carlsbad, Calif.) according to manufacturer's directions. qPCR isperformed in 25 μl total volume with 2×IQ SYBR Green supermix (SYBRGreen I dye, 50 U/ml iTaq DNA polymerase, 0.4 mM each of dATP, dCTP,dGTP and dTTP, 6 mM MgCl₂, 40 mM Tris-HCl, pH 8.4, 100 mM KCl, and 20 nMfluorescein (BioRad, Hercules, Calif.)), 300 nM final concentration eachfor forward and reverse primers listed in Table 1, sweet potato totalDNA (5-10 ng/reaction), and qPCR water (Ambion, Austin, Tex.) to finalvolume. qPCR amplifications are performed in a MyiQ (BioRad, Hercules,Calif.) thermal cycler with the following conditions: 95.0° C. for 3minutes; 40 cycles of 95.0° C. for 30 seconds, 60.0° C. for 30 seconds,72.0° C. for 30 seconds; with FAM channel optics “on” during annealingstage. Negative control is performed without any DNA (“no templatecontrol” or “NTC”), substituting same volume of molecular grade waterfor DNA. Positive and normalizing controls are used for all assays. Fora sample to be considered positive, its threshold cycle (Ct) value mustbe less than all negative control reactions, and the correspondingamplification curve has to exhibit the three distinct phases ofreal-time PCR: lag, linear and plateau.

All four primer sets produced amplicons of expected lengths (81 bp, 108bp, 141 bp, and 174 bp) when run in 1% agarose gels. All amplicons areisolated and sequenced, and each amplicon exhibits 100% identity to theIpomoea batatas atp1 mitochondrial gene under NCBI BLAST analysis aswell as atp1 in many other plants. The sequence of the 174 bp amplicongenerated by 174F and 174R primers is SEQ ID NO: 14. The sequence of the108 bp amplicon generated by 108F and 108R primers is SEQ ID NO: 15. Thesequence of the 81 bp amplicon generated by 81F and 81R primers is SEQID NO: 16. The sequence of the 141F and 141R primers is SEQ ID NO: 17.Of the four primer pairs assayed, primer set 174 exhibits the greatestdifference in Ct values between the two samples, autoclaved sweet potatoand unautoclaved control sweet potato (9 Ct difference versus 8, 5 & 5for amplicon lengths of 141, 108, and 81 base pairs, respectively). Thisresult is understandable as longer amplicons are statistically morelikely to experience degradation than shorter ones. As such, of thesefour sets of primer pairs, 174F and 174R, provides better resultscompared to the other three primer sets and, thus, is used in the otherexamples described infra. However, this invention is not limited to theabove listed primers. Any other primer set that has high identity toatp1 and which yields an amplicon of between approximately 80 bp toapproximately 250 bp can be used for fruits, vegetable, and nuts. Inanother embodiment, the primer set for assaying fruits, vegetable andnuts generates an amplicon within atp1 ranging from approximately 100 bpto approximately 200 bp. In yet another embodiment, the primer set forassaying fruits, vegetable and nuts generates an amplicon within atp1ranging from approximately 125 bp to approximately 175 bp. It is alsohelpful that the primer set used for any food matrix or medical devicegenerates a Ct difference of approximately 9 or greater between thenegative control (unprocessed sample) and processed sample.

Standard curves are generated using double-stranded, sequence-verifiedoligonucleotides of the I. batatas atp1, having the same sequence as theamplicon generated by the 174F and 174R primers (SEQ ID NO: 14)(gBlocks® gene fragments purchased from IDT (Coralville, Iowa)).Ten-fold serially dilutions of atp1 amplicon copies (10⁷ to 10¹) areperformed, qPCR is performed using the protocol above with 174F and 174Rprimers. PCR amplification efficiency (E) is determined using the slopeof the standard curve: E=(10^(−1/slope))−1. Data analysis of the qPCRstandard curve is performed using goodness-of-fit linear regressioncorrelation coefficient (R²). The slope value is used to assess therobustness of the assay using the efficiency value above. Theamplification efficiency is calculated as 106%, R²=0.9884 (see FIG. 1).

Bustin, et al. (2009) published the MIQE guidelines (Minimum Informationfor publication of Quantitative real-time PCR Experiments) to facilitateassessment and evaluation of new, clinical qPCR assays. The guidelinesinclude a checklist for authors, reviewers and editors to help themensure the integrity of scientific literature and promote consistencybetween laboratories (Bustin, et al. (2009)). According to theguidelines, essential information includes experimental design, sampledescription and processing, nucleic acid extraction, qPCR targetinformation, qPCR oligonucleotides, qPCR protocol, qPCR validation, anddata analysis. The present invention meets all essential informationrequirements.

Example 2 Hot Oil Bath as Substitute for Industrial Microwave System

A hot oil bath (EW-111, Neslab Instruments, Newington, N.H.) filled with8 L white mineral oil (Therminol XP, Solutia, Inc, St. Louis, Mo.) isused to maintain a temperature of 121.1° C. for substances placed in athermal death tube (TDT). A thermal death tube allows one to replicatethe conditions of an industrial microwave system yet still obtainsamples of the substance at various time points. 100 μl sweet potato(SP) puree in 1:4 dilution with 0.9% saline or 100 μl of Geobacillusstearothermophilus (GS) spores (ca. 10⁸ CFU/ml) are inserted intoseparate TDTs and are sealed according to instructions. Samples areheated for 0, 0.5, 1, 2, 4, 8, 16 and 20 minutes at 121.1° C., takinginto account the 30 seconds come up time (CUT). Three repetitions arerun per time point; three TDTs are placed in a metal tea strainer tofacilitate removal of samples from hot oil. Strainers containing TDTsare taken out of oil bath and immediately placed in an ice slurry for 30seconds to quickly cool them. Strainers are stored at room temperatureuntil ready for DNA extraction or culture plating. Total amount of sweetpotato puree recovered from hot oil bath treatment ranges betweenapproximately 50 μl to approximately 75 μl from an initial sample of 100μl (see FIG. 2).

GS spores are serially diluted with 0.9% saline solution and plated witha spiral plater (Spiral Biotech, Inc., Norwood, Mass.) on BHI agar(Becton Dickinson, Sparks, Md.). After 24 hours incubation at 55° C.,colonies are enumerated with an automated spiral plate counter (Q-count,Spiral Biotech Inc. Norwood, Mass.). The lower detection limit is 10²CFU/ml. Another G. stearothermophilus indicator system, the Prosporeampoule (Mesa Laboratories, Inc, Lakewood, Colo.) are incubated at 55°C. for 48 hours, and then checked for color change as directed.

Treated sweet potato puree is removed from TDT and placed directly intoa MO BIO bead beater tube (Carlsbad, Calif.). The MO BIO PowerSoil® DNAisolation kit (Carlsbad, Calif.) is used according to manufacturer'srecommendations to extract sweet potato DNA from the treated puree.Total DNA samples are analyzed by spectrophotometer (Nanodrop,Wilmington, Del.) for quantity and quality. For qPCR, DNA is normalizedby concentration: between 5-10 ng/μl per reaction.

qPCR is performed in 25 μl total volume with 2×IQ SYBR Green supermix(SYBR Green I dye, 50 U/ml iTaq DNA polymerase, 0.4 mM each of dATP,dCTP, dGTP and dTTP, 6 mM MgCl₂, 40 mM Tris-HCl, pH 8.4, 100 mM KCl, and20 nM fluorescein (BioRad, Hercules, Calif.)), 300 nM finalconcentration each for 174F and 174R primers, sweet potato DNA (5-10ng/reaction) and qPCR water (Ambion, Austin, Tex.) to final volume. qPCRamplifications are performed in a MyiQ (BioRad, Hercules, Calif.)thermal cycler with the following conditions: 95.0° C. for 3 minutes; 40cycles of 95.0° C. for 30 seconds, 60.0° C. for 30 seconds, 72.0° C. for30 seconds; with FAM channel optics “on” during annealing stage. Notemplate control (NTC) and positive controls are used for all assays. Apositive control is used to normalize data between assays. For a sampleto be considered positive, its threshold cycle (Ct) value must be lessthan all negative control reactions, and the corresponding amplificationcurve has to exhibit the three distinct phases of real-time PCR: lag,linear and plateau.

FIG. 2 illustrates the effect of hot oil bath (at 121° C.) on sweetpotato puree mtDNA fragmentation (increase in Ct value) over time. Inthe hot oil bath, Ct value increased from approximately 24 at time zeroto between approximately 30 and approximately 32, a 6-8 unit increase.This hot oil bath assay exhibits a high correlation (R²=0.97) betweensurrogate GS spore destruction and increase in Ct value over time (seeFIG. 3).

Example 3 Autoclave Degradation of Sweet Potato mtDNA and G.stearothermophilus Spores

To determine if mtDNA also fragments during the high heat and pressureof autoclaving, and assess the level of degradation of the DNA, sweetpotato puree and G. stearothermophilus spores are assayed in alaboratory autoclave (Amsco Eagle SG-3021 Scientific Gravity Sterilizer,Steris Corp., Mentor, Ohio). The autoclave is programmed to run liquidsterilizing times of 2, 4, 8 and 20 minutes at 121° C. Come up times(initial CUT=5 minutes, all others=1 minute) and come down times(Exhaust=11:53 to 13:25 minutes) are similar for all runs. In a plasticmicro-centrifuge holder three samples are included per run: 300 mg sweetpotato puree (prepared as described supra), 250 μl G. stearothermophilusspores (log 8 CFU/ml) (prepared as described supra) both samples in 1.5ml micro-centrifuge tubes containing a small hole in the top to ventwater vapor, and one commercial G. stearothermophilus vial (Prospore,Mesa Laboratories, Inc, Lakewood, Colo.). Vent holes are then coveredwith parafilm. After autoclave treatment, sample tubes are placed on iceuntil they cool to room temperature. DNA is extracted from each sampleusing the PowerSoil® DNA isolation kit (MO BIO, Carlsbad, Calif.) usingmanufacturer's recommendations. DNA is quantified and qualified viaspectrophotometry (Nanodrop, Wilmington, Del.). G. stearothermophilusspores are serially diluted as described supra and are plated with aspiral plater (Spiral Biotech, Inc., Norwood, Mass.) on BHI agar (BectonDickinson, Sparks, Md.). After 24 hours incubation at 55° C., coloniesare enumerated with an automated spiral plate counter (Q-count, SpiralBiotech, Inc., Norwood, Mass.). The lower detection limit is 10² CFU/ml.Prospore ampoules (Mesa Laboratories, Inc, Lakewood, Colo.) areincubated at 55° C. for 48 hours, and then checked for indicator colors.qPCR is performed using the above described methods.

In the autoclave treatment, Ct value for sweet potato puree mtDNAincreases from approximately 25 at time zero to 32 after 20 minutes. Ctvalue increase is similar in the autoclave treatment which is conductedusing the same time/temperature profile as the oil bath. The autoclaveassay exhibits a high correlation (R²=0.87) between surrogate G.stearothermophilus spore destruction and increase in Ct value over time(see FIG. 4)

Example 4 Assaying mtDNA Degradation on Various Heat Processed FoodSubstances

The atp1 gene is found to be highly conserved among plant species. Asdescribed above, BLAST analysis of the 174 forward and reverse primersreveals that they exhibit 100% identity with a wide variety of fruits,nuts and vegetables. To assess if the 174 primers can be useduniversally to test plant-based foods, both singly and in mixtures suchas soups, the following experiment is performed. Fresh, uncooked fruits,vegetables and nuts (see Table 2 for items) are purchased from a retailgrocery store. The samples are processed immediately by grinding in aHamilton Beach coffee mill. The coffee mill is thorough cleaned withdistilled water and 70% ethanol between samples and repetitions toprevent DNA cross-contamination. For each variety of plant tested, threeseparate samples of that plant are used. Six repetitions are tested inall, three uncooked controls and three autoclave treatments (20 minutesat 121° C. using procedure described above). The tissue culture protocolof the MasterPure DNA purification kit (Epicentre, Madison, Wis.) isused according to manufacturer's recommendations. DNA is quantified andqualified using a spectrophotometer (Nanodrop, Wilmington, Del.). DNA isnormalized to 5-10 ng/well and undergo qPCR assays using the 174F and174R primers and the protocol described above. Each sample is run induplicate wells. DNA is saved at −20° C. in case amplicon sequencing isrequired. Mean Ct values for uncooked and autoclaved plant materials arerecorded as well as the increase of Ct caused by autoclave treatment andthe slope of the line formed by the graph of the two values. The resultsare presented in Table 2.

TABLE 2 Mean Mean uncooked autoclaved Sample Ct Ct Difference SlopeVegetables White potato 19.73 32.77 13.04 0.65 Sweet potato 24.06 33.008.90 0.45 Tomato 18.86 32.27 13.41 0.67 Green pepper 19.45 35.66 16.210.81 Red pepper 18.99 34.63 15.65 0.78 Jalapeno 19.96 35.66 15.70 0.79pepper Carrot 15.71 32.45 16.74 0.84 Green bean 22.45 32.38 9.93 0.50Corn 22.40 27.24 4.84 0.24 Cucumber 18.47 29.88 11.40 0.57 BiofuelsSwitch grass 28.26 34.69 6.43 0.32 Fruits Apple 22.95 36.27 13.32 0.67Blueberry 25.88 35.51 9.63 0.48 Peach 20.93 37.52 16.60 0.83 Strawberry23.27 33.17 9.90 0.50 Pineapple 22.97 33.31 10.35 0.52 Grape 27.95 32.114.16 0.21 Nuts Peanut* 17.00 23.10 6.10 0.32 Almond 18.31 27.25 8.940.45 Pecan 25.86 31.43 5.57 0.28 *Roasted at 167° C. for 19 min. Allothers autoclaved at 121° C. for 20 min.

Using the 174F and 174R primers, a significant, detectable increase inCt value (approximately 4 to approximately 17 units) is found betweenuncooked and autoclaved samples (121° C. for 20 minutes) for thevegetables, fruits and nuts listed in Table 2. Therefore, the 174F and174R primers are be suitable for quantifying heat treatment efficacy andquality in a wide variety of plant foods, in complex mixtures such assoups, and plant precursors for biofuels. Not wishing to be bound to anyparticular hypothesis, Ct differences that occur between different foodsmay result from differences in DNA extraction because some plant foodsare high in sugars or fats, while other plant foods are high in fiber.

Example 5 mtDNA Degradation in Acidified Food Substances

Determining mtDNA fragmentation can assess shelf life in vegetableproducts. Cucumbers and hamburger dill chips are assayed duringprocessing and storage (FIG. 5). Cucumbers are fermented in NaCl for 8months, are pasteurized at 75° C. for 15 minutes and then are stored atroom temperature. Using the qPCR protocol and the 174F and 174R primers,both described supra, mtDNA fragmentation at pre-fermentation,immediately after pasteurization, at 2 months, and at 20 months isassayed. mtDNA fragmentation of autoclaved cucumbers is performed forcomparison using the protocols provided above. Threshold cycle (Ct)values are significantly different between all treatments, exceptbetween a fermented and pasteurized cucumber stored for 2 months and anautoclaved cucumber. There is a significant difference (student t-test,P<0.05) between the pickles stored at 2 and 20 months. The fermented,pasteurized pickle has a similar Ct value as the autoclaved cucumber.The results demonstrate that lower thermal processes (75° C. for 15minutes) under acidified conditions (pH=3.8) yield similar mtDNAfragmentation results to more elevated temperature conditions (121° C.)and that thermal processes under 100° C. with high acid products can bemonitored for the reliability of the heat treatment, acidificationand/or fermentation using qPCR of mtDNA.

Example 6 mtDNA Degradation in Dry Roasted Peanuts

To demonstrate that mtDNA degradation is an effective time/temperatureintegrator for roasted solid foodstuffs such as nuts, Virginia greenrunner peanuts are spiked with 10⁸ CFU/g Enterococcus faecium (ATCC8459; a Salmonella surrogate) and compared with Ct values of the samepeanut samples (not spiked with E. faecium) during dry roasting at 167°C. E. faecium is inoculated into BHI broth (Remel, Lenexa, Kans.) fromfreshly plated colonies and incubated statically overnight at 35° C.Cultures are concentrated 2× by centrifugation (5810R, Eppendorf,Hamburg, Germany) at 6,000 rpm for 10 minutes at 4° C. and resuspendedin 0.5× initial volume with sterile 0.9% saline. Target concentration is10⁸ CFU/ml. Initial culture concentration is determined by spectrometryat A600 and a simplified agar plate technique (Jett, et al.,BioTechniques 23:648-650 (1997)) utilizing square petri dishes and thetrack-dilution method. E. faecium-inoculated saline is added to thetotal weight of peanuts to be tested in a large plastic zipper bag,diluting the culture 1:20. The bag is closed and secured. Contents aremixed thoroughly, and then sit for 5 minutes to absorb the liquid.Inoculated peanuts are poured onto wire racks in 100 g aliquots andallowed to air dry for 20 minutes.

A convection oven (Despatch Model LXD1-42-2; Minneapolis, Minn.) is setto 167° C. and is allowed to equilibrate for 30 minutes. A metal rack isinserted in the oven and is brought to temperature. This rack holdssmaller racks made of hardware cloth through which peanuts do not pass.Each batch consists of 100 g of peanuts laid out on the small hardwarecloth racks. When the smaller racks are inserted into the larger rack,the peanuts are essentially suspended in the moving air inside the oven.For each roasting batch, the oven door is quickly opened, the tray withthe peanuts is slid into the large rack, and the door of the ovenquickly closed. There is a drop in the oven temperature caused by thedoor opening. The lowest temperature reached and the number of secondsrequired for the oven to return to set point are recorded for eachbatch. At the appropriate time point, the oven is opened quickly, andpeanuts are removed in the small rack. This rack is placed over ahomemade cooler with sufficient flow to cool the peanuts to roomtemperature in 30 seconds. To prevent cross contamination betweenrepetitions, a clean gloves are used for loading each batch into thesmall rack and into the oven, and clean gloves are used to remove eachbatch. Between runs, the small rack and the cooler are sprayed with 70%ethanol and allowed to dry thoroughly before the next batch comes intocontact with them. Cooled peanuts are placed in plastic bags and storeduntil ready for mtDNA analysis and E. faecium plate count analysis. Itis acknowledged that while the oven used for this experiment hasconvective airflow, the oven is not comparable to industrial-scale,commercial ovens used for peanuts. However, it is assumed that theresults would be similar when an industrial-scale, commercial oven isused.

Three replicate samples are taken from the following time points: 0, 3,6, 9, 12, 15, 18, and 21 minutes. Ten grams are taken from each 100 greplicate and placed in a stomacher bag (Filtra-bag, Fisher, Pittsburgh,Pa.) with 10 ml sterile 0.9% saline (1:1 dilution). Peanuts arestomached in a Seward Stomacher 400 (Tekmar, Cincinnati, Ohio) for 2minutes at normal speed. Filtrate is aseptically removed from thestomacher bag, serially diluted and plated as described above using thesimplified agar plate technique (Jett, et al. (1997)) with BHI agar (BD,Sparks, Md.). Plates are incubated at 35° C. over-night. Plates arecounted manually, and CFU/g peanuts are calculated, taking into accountconcentration and dilution factors.

Three peanuts from each replicate are ground under liquid nitrogen in amortar and pestle. The mortar and pestle are thoroughly cleaned betweensamples with 70% ethanol to prevent cross contamination. DNA isextracted using ca. 2.5 mg or one inoculation loop of ground peanut inthe MasterPure DNA purification kit (Epicentre, Madison, Wis.) using thetissue sample portion of the protocol. DNA is quantified and qualifiedwith a spectrophotometer (Nanodrop, Wilmington, Del.).

qPCR is performed in 25 μl total volume with 2× IQ SYBR Green supermix(SYBR Green I dye, 50 U/ml iTaq DNA polymerase, 0.4 mM each of dATP,dCTP, dGTP and dTTP, 6 mM MgCl₂, 40 mM Tris-HCl, pH 8.4, 100 mM KCl, and20 nM fluorescein (BioRad, Hercules, Calif.)), 300 nM finalconcentration each for 174 forward and 174 reverse primers, peanut DNA(5-10 ng/reaction) and qPCR water (Ambion, Austin, Tex.) to finalvolume. Amplifications are performed in a MyiQ (BioRad, Hercules,Calif.) thermal cycler with the following conditions: 95.0° C. for 3minutes; 40 cycles of 95.0° C. for 30 seconds, 60.0° C. for 30 seconds,72.0° C. for 30 seconds; with FAM channel optics “on” during annealingstage. No template control (NTC) and positive controls are used for allassays. The positive control is used to normalize data between assays.For a sample to be considered positive, its threshold cycle (Ct) valuemust be less than all negative control reactions, and its correspondingamplification curve must exhibit the three distinct phases of real-timePCR: lag, linear and plateau. Ct values are initially steady, thenincrease after 12 minutes of roasting to a mean value approximately 22units after 21 minutes (see FIG. 6). Peanut mtDNA fragmentation iscorrelated to E. faecium death because R²=0.67 (see FIG. 7).

The remainder of each batch of peanuts is used to determine the Hunter Lvalue color. The skins are removed from approximately 40 g peanuts whichare placed in a glass petri dish and inserted above a calibratedHunterLab DP9000 with D25 sensor (Hunter Associates Laboratory, Reston,Va.) utilizing the Lab scale (a standardized color scale). Readouts arerecorded three times per sample with the peanuts removed and resorted inthe petri dish between readings with the peanuts placed outer-side down,if broken. The color is expressed as the mean of the three replicationsfor each peanut sample presented to the colorimeter. The scale of thereadings range from 1 to 100 with 1 representing black and 100representing white. Peanut mtDNA fragmentation is a good correlation toHunter L color (see FIG. 8), a quality parameter used to determineroasting endpoint because R²=0.95.

Experiment 7 Using of Fluorescent Probe with Hot Oil Bath as Surrogatefor Industrial Microwave System

The protocol of Experiment 2 is repeated except instead of using SYBRGreen I dye (an intercalater of DNA), a nucleic acid probe such asTaqMan® is used to measure the amount of mtDNA degradation. TaqMan®probes contain a fluorescent reporter dye (e.g., 6-carboxyfluorescein(6-FAM™) or tetrachloro-6-carboxy-fluorescein (TET)) at the 5′ end and aquencher dye at the 3′ end (e.g., Iowa Black FQ or Black Hole Quencher(BHQ-2) quenchers). For TaqMan® detection, during each amplificationcycle the probe attaches along with the primers to the target sequenceof DNA to be copied. As the DNA strand is copied, the reporter dye isreleased from the probe sequence, and its fluorescent signal ismeasurable because it is no longer near the quencher dye. The amount offluorescence increases with each PCR cycle in proportion to the amountof target DNA amplified, thereby allowing direct detection andquantification of the target DNA sequence with a high degree ofspecificity, accuracy, and sensitivity. The probe's sequence can rangefrom approximately 15 bp to approximately 30 bp and is the sequence ofthe coding or non-coding strand (reverse complement of the coding strandsequence) of the amplicon.

The GS spores and sweet potato are processed according to the protocolin Experiment 2. The fluorescence of the samples is measured, and the Ctvalues obtained are almost identical to the values obtained inExperiment 2.

While the above experiments examined the Ct value of atp1 from mtDNA,one can determine the Ct value of any mtDNA. One can pick any primersthat generate a desired amplicon of approximately 250 bp or less duringthe DNA amplification step.

Example 8 Extrinsic DNA Thermometer Testing the Efficacy ofPasteurization and Other Thermal Processes

This experiment demonstrates the use of extrinsic DNA as atime/temperature indicator for inactivation of hazardous biologicalmaterial when one does not want or cannot perform qPCR on intrinsic DNA.One example of using this type of assay is to assess the efficacy ofinactivation of hazardous biological material on a food container (e.g.,a jar) or a medical device. Extrinsic DNA thermometers (described infra)are time/temperature indicators which are added and recovered fromthermal processing or other inactivation processing methods for foods,packaging, and medical equipment to test the efficacy of theinactivation system and/or method. A solution is prepared containing0.5% citric acid and divided into three aliquots. Each aliquot isadjusted to either pH 3.6, 4.0 or 4.4 using 1M NaOH. All solutions arefilter sterilized by passage through 0.45 um filters. An extrinsic DNAthermometer is created by combining 2 μl of 10⁸ copies/μl of the 174primer amplicon for atp1 (SEQ ID NO: 14) (gBlock® gene fragmentpurchased from IDT, Coralville, Iowa) (see FIG. 1) with 18 μl of acitric acid solution in a 200 μl domed thermal cycler tube. Finalconcentration of extrinsic DNA in each tube is 10⁷ copies/μl. Tubes areplaced in a thermal cycler (MyiQ, BioRad, Hercules, Calif.) when thetemperature reaches 96° C. Samples are removed at time points 0, 4, 8,16, 24 minutes in assay 1; and 0, 1, 2, 3, 4, 5 minutes in assay 2; withthree reps at each time point using three different pH levels. Totalnumber of samples is 45 each for each assay. After heat treatment,samples are placed in ice water slurry until cool, approximately 10minutes. Atp1 qPCR protocol is run on each sample as described suprausing 174F and 174R primers, being careful to segregate amplicon fromreagents and pipettemen. FIGS. 10 and 11 show Ct values of extrinsic DNAthermometer, consisting of gBlock® of atp1 amplicon (SEQ ID NO: 14) in0.5% citric acid, versus time at 96° C. FIG. 10 runs from 0-24 minutes,FIG. 11 from 0-5 minutes. For longer time courses, pH 4.4 had the bestgoodness-of-fit value (FIG. 10: R²=0.90). For shorter thermal processes,5 minutes or less at 96° C., pH 4.4 had the best goodness-of-fit (FIG.11; R²=0.67). Extrinsic DNA thermometers can be used in lowtemperature/low acid thermal processes, medical or containerapplications, or any process where intrinsic DNA is difficult to obtain.

While a citric acid solution to hold the extrinsic DNA is used in thisexample, one can use any organic acid or inorganic acids to generate alow pH solution into which extrinsic DNA is placed. A non-limitingexample of an organic acid is malic acid. Non-limiting examples ofinorganic acids are HCl and phosphoric acid. Further, while this exampleused atp1 amplicon having SEQ ID NO: 14, any double stranded DNA ofbetween approximately 80 bp and approximately 250 bp (contiguous bp) ofatp1 or any mitochondrial DNA can be used with the appropriate primersto generate an amplicon of the sequence used. Also a labeled probe canbe used into of an intercalating dye as per the above protocol.

One can use extrinsic DNA to determine the efficacy of inactivation of ahazardous biological material in a food matrix, instead of analyzingintrinsic DNA. One simply needs to submit an amount of extrinsic DNA tothe processing methods of the food matrix and then determine the Ct ofthe extrinsic DNA. The extrinsic DNA may need to be placed in acontainer.

Example 9 Using Total DNA Fragmentation of Plant Food Matrix DuringThermal Processing as a Measure of Processing Efficacy

DNA samples from previous described assays and similar concentrations(approximately 200 ng/μl) are loaded into a mini electrophoretic unitcontaining a global DNA analyzer (Agilent Bioanalyzer 2100, Santa Clara,Calif.). The analyzer either contains a fluorescent composition thatbinds to the DNA within the electrophoretic gel or one adds it to theanalyzer. A graph comparing DNA fragment size (ranging fromapproximately 35 to approximately 10,380 bp) with fragment concentrationis generated. Graphs of total DNA size from fresh and autoclavedcucumber DNA are compared. FIG. 12 illustrates the global measurement oftotal cucumber DNA before and after autoclave treatment. Number of basepairs (size) versus concentration (pg/ul) are compared. Fresh cucumberDNA ranges from <1,000 base pairs (bp) to approximately 10,000 bp. Afterautoclave treatment, total DNA is degraded and fragmented (<3,500 bp),sizes clustering between approximately 35 bp and approximately 400 bp.DNA integrity is the wholeness or completeness of a cell's genomic andorganelle-based DNA. After heat treatments such as autoclaving, a cell'sDNA integrity is reduced and this fragmentation can be measuredglobally. Based on the results, an algorithm which predictstime/temperature treatment based on global DNA fragmentation isgenerated. One uses can use this measurement of DNA integrity as atime/temperature integrator for inactivation of hazardous biologicalmaterial in/on food matrices and/or medical devices.

Again, one can use extrinsic DNA or intrinsic DNA with this protocol foranalyzing the DNA fragmentation.

A flow chart illustrating the methods of these novel time/temperatureintegrators of these inventions is in FIG. 13.

Example 10 Comparison of Ct and F Values of Sweet Potato Puree with12D-Retort Protocol

Sweet potato puree is produced as described supra and placed in68.3×101.6 mm cans outfitted with T-type C-2 tube and rod thermocouples(Ecklund-Harrison Technologies, Fort Myers, Fla.). Colorimetric G.stearothermophilus ampoules (Raven ProSpore; Mesa Laboratories, Inc.,Lakewood, Colo.) are placed in the center of each can, adjacent to thethermocouple probes. Cans are sealed with a double seam using anautomated can sealer (Dixie Canner Co., Athens, Ga.). Total weights ofpuree and size of head space are similar between all cans in each run.Canned sweet potato puree is loaded into a Model PR-900 pilot retort(Stock sterilisationstechnik, Hermanstock Maschf.; Neumunster, WestGermany) with thermocouples attached to a recording device and run inone of two full water immersion protocols listed in Tables 3 and 4infra. Protocol 00 (Table 3), sub-12D full water immersion, is asubstandard treatment not meant to kill spores. Protocol 01 (Table 4),12D full water immersion, is a 12D protocol meant to eliminate all G.stearothermophilus test spores. Each protocol is run in triplicate usingthree cans per run. Puree is sampled from the center of each can, theDNA is extracted, and the atp1 qPCR protocol performed as describedsupra using primers 174F and 174R. ProSpore ampoules are incubated at55° C. for 48 hours as recommended by the supplier, and then assessedfor colorimetric change. F values are determined from the thermal coupletime-temperature data collected. F value is calculated as follows:F=10^((T-121.1/10)) Δt; where T is temperature in ° C. and t is time inminutes. Ct values are correlated to F values of all 12D and sub-12Druns and are shown in FIG. 14A (protocol 00 which is shown in Table 3)and FIG. 14B (protocol 01 which is shown in Table 4). The protocolspresented in Table 3 and Table 4 contain information provided by themanufacturer (Hermanstock Maschf.; Neumunster, West Germany). Theresults support the use of qPCR of mtDNA of a food product to assessbacterial spore inactivation, because the Ct values are highlycorrelated to F values.

TABLE 3 Temperature Pressure Temp Pressure Step (F.) (psi) Time GradientGradient Heating 180 20 — — — Storage Vessel Sterilization I 180 20 20sec — — (Vent) Sterilization II 242 20 10 min 10 — (Come up)Sterilization III 242 20 35 min — — (Hold) Cooling 1 — 20 10 min — —Cooling 2 90 10 20 min — 0.6 Drain 90 —  4 min — —

TABLE 4 Temperature Pressure Temp Pressure Step (F.) (psi) Time GradientGradient Heating 200 20 — — — Storage Vessel Sterilization I 200 20 20sec — (Vent) Sterilization II 260 20 12 min 10 — (Come up) SterilizationIII 260 20 35 min — — (Hold) Cooling 1 — 20 10 min — — Cooling 2 90 1012 min — 0.6 Drain 90 —  4 min — —

Example 11 Comparison of Ct Value with D- and z-Values

In an effort to mimic and quantify values in a 12D thermal process, thekill curve of G. stearothermophilus (a C. botulinum surrogate) sporeswith resulting D- and z-values are compared to Ct values of sweet potatopuree in a hot oil bath at the following temperatures: 116° C., 121° C.,123° C., and 126° C. A hot oil bath (Neslab Instruments, Newington,N.H.) filled with 8 L white mineral oil (Solutia, Inc, St. Louis, Mo.)is used to maintain each target temperature for substances placed in athermal death tube (TDT). This system replicates conditions in anindustrial retort, heat exchanger or microwave thermal process. The TDTis composed of a ¾ inch aluminum screw post (Screwpost.com, Muskegon,Mich.) cut to size and filed for smoothness, ¼ inch nylon machinescrews, Viton fluoroelastomer O-ring gaskets (screw size #6) and Vitonflat washers size #6 (McMaster-Carr, Atlanta, Ga.). Temperature ismonitored using a type J-K-T microprocessor thermometer thermocouple(Omega Corp., Stamford, Conn.). Come up time (CUT) for TDTs isdetermined for all target temperatures. In each TDT, 100 μl of 1:4diluted puree or 100 μl of G. stearothermophilus (GS) spores(approximately 10⁸ CFU/ml) are inserted and sealed. For GS spores,samples are heated for 0, 4, 8 and 12 minutes at 116° C.; for 0, 0.5, 1,2, 4, 8, 16 and 20 minutes at 121° C.; for 0, 1, 2 and 4 minutes at 123°C.; and for 0, 0.5, 1, 2 and 4 minutes at 126° C., with all heattreatments beginning after come up time. Samples of diluted sweet potatopuree are heated for 0, 12, 24, 48 and 60 minutes at 116° C.; for 0, 4,8, 12 and 18 minutes at 121° C.; for 0, 4, 8, 12 and 18 minutes at 123°C.; and for 0, 4, 8, 12 and 18 minutes at 126° C.; with all heattreatments beginning after come up time. Three replications are run pertime point; 3 TDTs are placed in a metal tea strainer to facilitateremoval of samples from hot oil. Strainers containing TDTs are taken outof oil bath and immediately placed in an ice slurry for 30 second toquickly cool them. Strainers containing TDTs are stored at roomtemperature until ready for DNA extraction or culture plating. Totalamount of sweet potato puree recovered from hot oil bath treatment isdetermined from an initial sample of 100 μl.

The D value (decimal reduction time) is defined as the time in minutesat a given temperature that results in a one log reduction in microbialcount (Pflug, Irving, Microbiology and Engineering of SterilizationProcesses. 7th Ed., published by Environmental Sterilization Laboratory;Minneapolis, Minn. (1990)). Using the equation:

N=N ₀10^(−t/D) _(T)

where N₀ and N are the initial and final number of microorganisms,respectively; the D value at a given temperature (D_(T)) is calculatedby graphing the log₁₀ number of microorganisms over time (minutes) anddetermining the slope: slope=−1/D_(T).

The z-value is the temperature change required for a one log change inthe D value of a microorganism (Pflug, Irvine supra (1990)). Using theequation

D _(T) =D _(ref)10^(Tref-T/z)

the z-value is calculated by graphing log D value (seconds) versustemperature and determining the slope: slope=−1/z.

G. stearothermophilus spores are serially diluted and plated with on BHIagar (Becton Dickinson, Sparks, Md.) using a spiral plater (SpiralBiotech Inc., Norwood, Mass.) or a simplified agar plate technique(Jett, et al., BioTechniques 23:648-650 (1997)). After 24 hoursincubation at 55° C., colonies are enumerated with an automated spiralplate counter (Spiral Biotech Inc., Norwood, Mass.) or counted manually.The lower detection limits are 4×10² and 1×10³ CFU/ml for the spiralplate and simplified agar technique, respectively.

Ct values are converted to log₁₀ copy numbers using the linearrelationship determined empirically from the standard curve of the 174bp amplicon:

y=−3.1909x+38.091

where y is the Ct value and x is the log₁₀ copy number.

D₁₂₁ and z-values determined in hot oil bath for G. stearothermophilusspores are 2.71 minutes and 11.0° C. (see FIGS. 15 and 16),respectively. These values are slightly higher than values obtainedusing a commercial product with the same GS strain spores for autoclavevalidation (Prospore, Mesa Labs, Lakewood, Colo.) which cite a D₁₂₁value of 1.8 minutes and a z-value of 7.4° C. under saturated steam.Prior art D and z-values for GS spores are Duo from 1.5 to 3 minuteswith z-value of greater than or equal to 6° C. and D₁₂₁ of approximately2 minutes in water (Lundahl, G., PDA J. Pharm. Sci. Technol. 57:249-262(2003)). Both of these cited values are based on an initial populationof 10⁶ spores. Head, et al. (J. Appl. Microbiol. 104:1213-1220 (2007))determined that D and z-values vary widely based on the initialconcentration of spores (10³ versus 10⁶) when treated with superheatedsteam. While the TDT employed herein is a pressurized container, onewould not expect the same time-temperature treatment in a hot oil bathas pressurized, saturated stream in an autoclave. Based on precautionarynotes in commercial spore technical data sheets (Prospore, Namsa,Northwood, Ohio) and values in the literature, spore D and z-values canvary widely because of the type of heat treatment (wet versus dry),initial concentration of spores, and spore carrier or media (Head, etal. (2007)). As an added precaution, a safety factor is added toempirically derived data, i.e. total death time is rounded up, to ensurecomplete destruction of spores (Tucker, et al., History of the minimumbotulinum cook for low-acid canned foods. Campden & Chorleywood FoodResearch Association Group. R & D report no. 260. DocRef:FMT/REP/90194/1 (2008)).

D₁₂₁ and z-values for Ct values from a 174-bp universal plant ampliconare 11.3 minutes and 17.8° C. (see FIGS. 17 and 18), respectively, formtDNA from sweet potato puree heated in a hot oil bath. The conversionof Ct to log₁₀ copy number of amplicon results in the Ct-D₁₂₁ value(11.3 minutes) being much higher than the G. stearothermophilus D121(2.71 minutes). G. stearothermophilus spores have a D121 valueapproximately 10× greater than C. botulinum (D₁₂₁=0.21 minutes; Esty &Meyer, J. Infectious Dis. 31:650-663 (1922); Townsend, et al., Food Res.3:323-346 (1938); Stumbo, C. R., Thermobacteriology in Food Processing.1^(st) Ed. Academic Press 111 Fifth Ave, New York, N.Y. (1965)), thespore of concern in low acid, canned or aseptically-packaged foods. TheCt-D₁₂₁ value of sweet potato puree mtDNA is approximately 4× greaterthan the G. stearothermophilus indicator spore. Because of its higherD121 value, it might be difficult to predict the FDA recommended F valuefor sterilization (F₀=5 minutes) using a log function of Ct value.However, sterilization in the pharmaceutical industry requires highervalues (F₀>12 minutes) where GS spores leave no measurable outcome(Lundahl, G. (2003)).

When compared directly, the increase in Ct value has nearly a 1:1 ratiowith G. stearothermophilus destruction at 121° C. in hot oil bathtreatments (ratio=0.875) (FIG. 19). The destruction of mtDNA as measuredby log₁₀ copy number is not a first order relationship but a simpleinverse relationship with time-temperature. Therefore, the use of Ctvalues directly will have greater utility than conversion to log values.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Alldocuments cited herein are incorporated by reference.

We, the inventors, claim:
 1. A kit comprising: a pair of primerscomprising a first polynucleotide and a second polynucleotide; a label;optionally DNase; and optionally instructions on using said firstpolynucleotide and said second polynucleotide in a method to determinethe amount of inactivation of a biological material, wherein said firstpolynucleotide has the sequence in one of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, and SEQ ID NO: 7, and wherein when said firstpolynucleotide has the sequence in SEQ ID NO: 1, said secondpolynucleotide has the sequence in SEQ ID NO: 2; when said firstpolynucleotide has the sequence in SEQ ID NO: 3, said secondpolynucleotide has the sequence in SEQ ID NO: 4; when said firstpolynucleotide has the sequence in SEQ ID NO: 5, then said secondpolynucleotide has the sequence in SEQ ID NO: 6; and when said firstpolynucleotide has the sequence in SEQ ID NO: 7, then said secondpolynucleotide has the sequence in SEQ ID NO:
 8. 2. The kit of claim 1wherein said label is selected from the group consisting of afluorescent composition, an intercalating dye, a spectroscopic label, aphotochemical label, a biochemical label, an immunochemical label, and achemical label.
 3. The kit of claim 1 wherein said label comprises: aprobe; a quencher dye; and a fluorescent dye, wherein said quencher dyeand said fluorescent dye are linked to said probe, wherein when thesequence of said first polynucleotide is SEQ ID NO: 1, said probe has asequence of between approximately 15 contiguous bases and approximately45 contiguous bases of SEQ ID NO: 14 or the reverse complement thereof,wherein when the sequence of said first polynucleotide is SEQ ID NO: 3,said probe has a sequence of between approximately 15 contiguous basesand approximately 45 contiguous bases of SEQ ID NO: 15 or the reversecomplement thereof, wherein when the sequence of said firstpolynucleotide is SEQ ID NO: 5, said probe has a sequence of betweenapproximately 15 contiguous bases and approximately 45 contiguous basesof SEQ ID NO: 16 or the reverse complement thereof, and wherein when thesequence of said first polynucleotide is SEQ ID NO: 7, said probe has asequence of between approximately 15 contiguous bases and approximately45 contiguous bases of SEQ ID NO: 17 or the reverse complement thereof.4. A kit comprising: a pair of primers comprising a first polynucleotideand a second polynucleotide; a label; optionally DNase; and instructionson using said first polynucleotide and said second polynucleotide in amethod to determine the amount of inactivation of a biological material,wherein said first polynucleotide has the sequence in one of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and wherein when saidfirst polynucleotide has the sequence in SEQ ID NO: 1, said secondpolynucleotide has the sequence in SEQ ID NO: 2; when said firstpolynucleotide has the sequence in SEQ ID NO: 3, said secondpolynucleotide has the sequence in SEQ ID NO: 4; when said firstpolynucleotide has the sequence in SEQ ID NO: 5, then said secondpolynucleotide has the sequence in SEQ ID NO: 6; and when said firstpolynucleotide has the sequence in SEQ ID NO: 7, then said secondpolynucleotide has the sequence in SEQ ID NO: 8.